Anti-Tumor Antigen Antibodies and Methods of Use

ABSTRACT

Antibodies that bind to tumor associated antigen CD44 or to tumor associated antigen EphA2, are disclosed herein, as well as related compositions and methods of use. Methods of use encompass cancer therapies, diagnostics, and screening methods.

CROSS-REFERENCE

This application claims the benefit of U.S. Provisional PatentApplication No. 61/366,823, filed Jul. 22, 2010, which application isincorporated herein by reference in its entirety.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with government support under grant no. CA058207awarded by the National Institutes of Health. The government has certainrights in the invention.

INTRODUCTION

Extensive studies of cancer transcriptional patterns have led to thediscovery of molecular targets to distinguish the malignant from thebenign and the most aggressive cancers from those that are lessaggressive. Cancers often overexpress a number of proteins, includingcertain cell surface antigens, e.g., cell surface receptors. Antibodiesthat bind such overexpressed cell surface antigens can facilitatedetection and treatment of such cancers. A number of approaches havebeen utilized to generate antibodies to cancer cell surface receptorswhich can be used as potential therapeutics. Identification ofoverexpressed cell surface receptors and antibodies which bind themprovide a route to the development of cancer therapies, especially forthose cancer subtypes with poor prognosis and resistance to traditionaltherapies. CD44 and EphA2 are two such overexpressed cell surfacereceptors, and are known from transcriptional profiling and proteomicanalysis to be overexpressed in basal breast cancers.

Antibodies specific to cell surface receptors overexpressed on a numberof cancers have been utilized for development of targetedimmunotherapeutics. For example, HER2, CD20, and EGFR are overexpressedon a number of tumors and antibodies recognizing these receptors havebeen developed to treat metastatic breast cancer (trastuzamab), lymphoma(rituximab), and colorectal cancer (cetuximab). Several therapeuticapproaches, including antibody-drug conjugates, immunotoxins, andtargeted nucleic acid delivery, require antibodies that not only bindreceptor, but that also undergo internalization into the cell uponbinding.

SUMMARY

Antibodies that bind to tumor associated antigen CD44 or to tumorassociated antigen EphA2, are disclosed herein, as well as relatedcompositions and methods of use. Methods of use encompass cancertherapies, diagnostics, and screening methods. In certain embodiments,antibodies bind mammalian cell surface antigen (e.g., yeast displayedmammalian cell surface antigen), in others they are endocytosed uponbinding to mammalian cells.

In one embodiment, an isolated monoclonal antibody is provided thatspecifically binds an epitope of CD44 that is specifically bound byantibody F2-1A6 or that competes with antibody F2-1A6 for binding toCD44. Said antibody may, when bound to CD44 on the surface of a livingmammalian cell, be endocytosed (by the cell). In one embodiment, saidantibody comprises a V_(H) CDR1 of F2-1A6, a V_(H) CDR2 of F2-1A6 and aV_(H) CDR3 of F2-1A6. In another embodiment, said antibody comprises aV_(L) CDR1 of F2-1A6, a V_(L) CDR2 of F2-1A6, and a V_(L) CDR3 ofF2-1A6. In yet another embodiment, said antibody competes for binding toan epitope of CD44 with an antibody comprising, a full length V_(H) ofF2-1A6, and a full length V_(L) of F2-1A6.

In another embodiment, an isolated monoclonal antibody is provided thatspecifically binds an epitope of CD44 that is specifically bound byantibody F2-1H9 or that competes with antibody F2-1H9 for binding toCD44. Said antibody may, when bound to CD44 on the surface of a livingmammalian cell, be endocytosed. In one embodiment, said antibodycomprises a V_(H) CDR1 of F2-1H9, a V_(H) CDR2 of F2-1H9, and a V_(H)CDR3 of F2-1H9. In another embodiment, said antibody comprises a V_(L)CDR1 of F2-1H9, a V_(L) CDR2 of F2-1H9, and a V_(L) CDR3 of F2-1H9. Inyet another embodiment, said antibody comprises a full length V_(H) ofF2-1H9 and a full length V_(L) of F2-1H9.

In another embodiment, an isolated monoclonal antibody is provided thatspecifically binds an epitope of CD44 that is specifically bound by anantibody selected from E8H11, E8H7, E8G12, E8F11, E8C9, D6G9, D6D3,D1C5, D1D1, HB8, HC2, HC4, HE3, HF1, and HH3; or that competes with anantibody selected from E8H11, E8H7, E8G12, E8F11, E8C9, D6G9, D6D3,D1C5, D1D1, HB8, HC2, HC4, HE3, HF1, and HH3 for binding to CD44. In oneembodiment, said antibody comprises: a V_(H) CDR1 of E8H11, E8H7, E8G12,E8F11, E8C9, D6G9, D6D3, D1C5, D1D1, HB8, HC2, HC4, HE3, HF1, or HH3; aV_(H) CDR2 of E8H11, E8H7, E8G12, E8F11, E8C9, D6G9, D6D3, D1C5, D1D1,HB8, HC2, HC4, HE3, HF1, or HH3; and a V_(H) CDR3 of E8H11, E8H7, E8G12,E8F11, E8C9, D6G9, D6D3, D1C5, D1D1, HB8, HC2, HC4, HE3, HF1, or HH3. Inanother embodiment said antibody comprises: a V_(L) CDR1 of E8H11, E8H7,E8G12, E8F11, E8C9, D6G9, D6D3, D1C5, D1D1, HB8, HC2, HC4, HE3, HF1, orHH3; a V_(L) CDR2 of E8H11, E8H7, E8G12, E8F11, E8C9, D6G9, D6D3, D1C5,D1D1, HB8, HC2, HC4, HE3, HF1, or HH3; and a V_(L) CDR3 of E8H11, E8H7,E8G12, E8F11, E8C9, D6G9, D6D3, D1C5, D1D1, HB8, HC2, HC4, HE3, HF1, orHH3. In another embodiment said antibody comprises: a full length V_(H)of E8H11, E8H7, E8G12, E8F11, E8C9, D6G9, D6D3, D1C5, D1D1, HB8, HC2,HC4, HE3, HF1, or HH3; and a full length V_(L) of E8H11, E8H7, E8G12,E8F11, E8C9, D6G9, D6D3, D1C5, D1D1, HB8, HC2, HC4, HE3, HF1, or HH3.

In another embodiment, an isolated monoclonal antibody is provided thatspecifically binds an epitope of EphA2 that is specifically bound byantibody 2D6 or that competes with antibody 2D6 for binding to EphA2.Said antibody may, when bound to EphA2 on the surface of a livingmammalian cell, be endocytosed.

In another embodiment, an isolated monoclonal antibody is provided thatspecifically binds an epitope of EphA2 that is specifically bound byantibody D2-1A7 or that competes with antibody D2-1A7 for binding toEphA2. Said antibody may, when bound to EphA2 on the surface of a livingmammalian cell, be endocytosed.

In another embodiment, an isolated monoclonal antibody is provided thatspecifically binds an epitope of EphA2 that is specifically bound byantibody D2-1A9 or that competes with antibody D2-1A9 for binding toEphA2. Said antibody may, when bound to EphA2 on the surface of a livingmammalian cell, be endocytosed.

In another aspect, an isolated monoclonal antibody is provided thatspecifically binds an epitope of EphA2 that is specifically bound byantibody D2-1B1 or that competes with antibody D2-1B1 for binding toEphA2. Said antibody may, when bound to EphA2 on the surface of a livingmammalian cell, be endocytosed.

In another embodiment, an isolated monoclonal antibody is provided thatspecifically binds an epitope of EphA2 that is specifically bound by anantibody selected from A3H9, A3G3, A3D10, A3D1, A3C8, 1A3, 1A5, 1A8,1A12, 1B2, 1C2, 1C7, 1D8, and 15H11 or that competes with an antibodyselected from A3H9, A3G3, A3D10, A3D1, A3C8, 1A3, 1A5, 1A8, 1A12, 1B2,1C2, 1C7, 1D8, and 15H11 for binding to EphA2. In one embodiment, saidantibody comprises: a V_(H) CDR1 of A3H9, A3G3, A3D10, A3D1, A3C8, 1A3,1A5, 1A8, 1A12, 1B2, 1C2, 1C7, 1D8, or 15H11; a V_(H) CDR2 of A3H9,A3G3, A3D10, A3D1, A3C8, 1A3, 1A5, 1A8, 1A12, 1B2, 1C2, 1C7, 1D8, or15H11; and a V_(H) CDR3 of A3H9, A3G3, A3D10, A3D1, A3C8, 1A3, 1A5, 1A8,1A12, 1B2, 1C2, 1C7, 1D8, or 15H11. In another embodiment, said antibodycomprises: a V_(L) CDR1 of A3H9, A3G3, A3D10, A3D1, A3C8, 1A3, 1A5, 1A8,1A12, 1B2, 1C2, 1C7, 1D8, or 15H11; a V_(L) CDR2 of A3H9, A3G3, A3D10,A3D1, A3C8, 1A3, 1A5, 1A8, 1A12, 1B2, 1C2, 1C7, 1D8, or 15H11; and aV_(L) CDR3 of A3H9, A3G3, A3D10, A3D1, A3C8, 1A3, 1A5, 1A8, 1A12, 1B2,1C2, 1C7, 1D8, or 15H11.

In certain embodiments, any of the aforementioned monoclonal antibodiesis a single chain Fv (scFv), IgG, Fab, (Fab′)₂, or (scFv′)₂. Any of saidantibodies may be labeled and/or be conjugated to an anti-cancer agent.

In one embodiment, a lipidic nanoparticle comprising a surface and aninterior space is provided, said interior space comprising ananti-cancer agent, wherein any one or more of the aforementionedisolated antibodies is attached to the surface of said lipidicnanoparticle. In one embodiment, when the lipidic nanoparticle iscontacted with a cell expressing cell surface EphA2 or cell surfaceCD44, said antibody binds to the cell surface EphA2 or cell surface CD44and the lipidic nanoparticle is endocytosed.

In one embodiment, a composition is provided comprising apharmaceutically acceptable carrier and any one of the aforementionedantibodies or any one of the aforementioned lipidic nanoparticles. Saidcomposition may be formulated for parenteral administration.Alternatively, said composition may be formulated for intravenous,intrathecal, or intraventricular administration or for convectionenhanced delivery. In one embodiment, a kit comprising the compositionis provided.

In another embodiment, a method of treating a subject having cancer isprovided, said method comprising administering to said subject an amountof any one of the aforementioned antibodies or any one of theaforementioned lipidic nanoparticles, wherein said amount is sufficientto slow the growth of the cancer. In one embodiment said antibody isinternalized into a cancer cell

In one embodiment, a method of detecting a cancer cell in a subject isprovided, comprising contacting an antibody of any one of theaforementioned antibodies with a cell of said subject suspected of beingcancerous and detecting said antibody bound to said cell.

In another embodiment, an isolated nucleic acid is provided, comprisinga nucleotide sequence encoding an amino acid sequence of: a V_(H)comprising a V_(H) CDR1, a V_(H) CDR2 and a V_(H) CDR3 of an antibodyfrom clone F2-1A6; a V_(L) comprising a V_(L) CDR1, a V_(L) CDR2 and aV_(L) CDR3 of an antibody from clone F2-1A6; a V_(H) comprising a V_(H)CDR1, a V_(H) CDR2 and a V_(H) CDR3 of an antibody from clone F2-1H9; aV_(L) comprising a V_(L) CDR1, a V_(L) CDR2 and a V_(L) CDR3 of anantibody from clone F2-1H9; a V_(H) comprising a V_(H) CDR1, a V_(H)CDR2 and a V_(H) CDR3 of an antibody selected from E8H11, E8H7, E8G12,E8F11, E8C9, D6G9, D6D3, D1C5, D1D1, HB8, HC2, HC4, HE3, HF1, and HH3;or a V_(L) comprising a V_(L) CDR1, a V_(L) CDR2 and a V_(L) CDR3 of anantibody selected from E8H11, E8H7, E8G12, E8F11, E8C9, D6G9, D6D3,D1C5, D1D1, HB8, HC2, HC4, HE3, HF1, and HH3. In one embodiment, saidnucleic acid comprises a nucleotide sequence encoding an amino acidsequence of: a V_(H) comprising a V_(H) CDR1, a V_(H) CDR2 and a V_(H)CDR3 of an antibody from clone F2-1A6 and a V_(L) comprising a V_(L)CDR1, a V_(L) CDR2 and a V_(L) CDR3 of an antibody from clone F2-1A6; aV_(H) comprising a V_(H) CDR1, a V_(H) CDR2 and a V_(H) CDR3 of anantibody from clone F2-1H9 and a V_(L) comprising a V_(L) CDR1, a V_(L)CDR2 and a V_(L) CDR3 of an antibody from clone F2-1H9; or a V_(H)comprising a V_(H) CDR1, a V_(H) CDR2 and a V_(H) CDR3 of an antibodyselected from E8H11, E8H7, E8G12, E8F11, E8C9, D6G9, D6D3, D1C5, D1D1,HB8, HC2, HC4, HE3, HF1, and HH3 and a V_(L) comprising a V_(L) CDR1, aV_(L) CDR2 and a V_(L) CDR3 of an antibody selected from E8H11, E8H7,E8G12, E8F11, E8C9, D6G9, D6D3, D1C5, D1D1, HB8, HC2, HC4, HE3, HF1, andHH3.

In another embodiment, an isolated nucleic acid is provided, comprisinga nucleotide sequence encoding an amino acid sequence of: a V_(H)comprising a V_(H) CDR1, a V_(H) CDR2 and a V_(H) CDR3 of an antibodyfrom clone 2D6, a V_(L) comprising a V_(L) CDR1, a V_(L) CDR2 and aV_(L) CDR3 of an antibody from clone 2D6, a V_(H) comprising a V_(H)CDR1, a V_(H) CDR2 and a V_(H) CDR3 of an antibody from clone D2-1A7, aV_(L) comprising a V_(L) CDR1, a V_(L) CDR2 and a V_(L) CDR3 of anantibody from clone D2-1A7, a V_(H) comprising a V_(H) CDR1, a V_(H)CDR2 and a V_(H) CDR3 of an antibody from clone D2-1A9, a V_(L)comprising a V_(L) CDR1, a V_(L) CDR2 and a V_(L) CDR3 of an antibodyfrom clone D2-1A9, a V_(H) comprising a V_(H) CDR1, a V_(H) CDR2 and aV_(H) CDR3 of an antibody from clone D2-1B1, a V_(L) comprising a V_(L)CDR1, a V_(L) CDR2 and a V_(L) CDR3 of an antibody from clone D2-1B1; aV_(H) comprising a V_(H) CDR1, a V_(H) CDR2 and a V_(H) CDR3 of anantibody selected from A3H9, A3G3, A3D10, A3D1, A3C8, 1A3, 1A5, 1A8,1A12, 1B2, 1C2, 1C7, 1D8, and 15H11; a V_(L) comprising a V_(L) CDR1, aV_(L) CDR2 and a V_(L) CDR3 of an antibody selected from A3H9, A3G3,A3D10, A3D1, A3C8, 1A3, 1A5, 1A8, 1A12, 1B2, 1C2, 1C7, 1D8, and 15H11;or an isolated nucleic acid comprising nucleotide sequences encoding theamino acid sequences of 1) and 2); 3) and 4); 5) and 6); 7) and 8); or9) and 10).

In one embodiment, an expression vector is provided that comprises anyof the aforementioned nucleic acids. In another embodiment, arecombinant host cell (a “genetically modified host cell”) is providedcomprising said vector. In one embodiment the cell expresses ananti-EphA2 antibody or an anti-CD44 antibody.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Display of antigen domains on the surface of yeast. Panel A, Theextracellular domain (ECD) of receptor EphA2 was displayed on the yeastsurface and was recognized by an anti-EphA2 antibody and recombinantmouse Ephrin A1 (R&D, Minneapolis) as determined by flow cytometryanalysis. Panel B, The link domain of CD44 (domain 1, or D1) wasdisplayed on the yeast surface and recognized by an anti-CD44 rabbitmonoclonal antibody as determined by flow cytometry analysis. Bothanti-EphA2 and anti-CD44 antibodies did not recognize an irrelevantprotein displayed on the yeast surface.

FIG. 2. Recovery of phage antibodies from yeast displayed antigens.Panel A, Comparison of the recovery of a phage antibody known to bindEphA2 (2D6) from yeast displaying EphA2 ECD (Y-EphA2) versus yeastdisplaying an irrelevant protein (Y-CON). A total of 10¹¹ anti-EphA2phage antibody 2D6 were incubated with each of the yeast displayedantigens. Panel B, Impact of the elution buffer on the titer of EphA2phage antibody eluted from the surface of yeast displaying EphA2 ECD. Atotal of 10¹¹ anti-EphA2 phage antibody 2D6 were incubated with each ofthe yeast displayed antigen proteins prior to elution. Panel C, Theimpact of input phage titer on the eluted phage titer. The indicatedtiter of anti-EphA2 phage antibody 2D6 was incubated with yeastdisplayed EphA2 ECD or an irrelevant yeast displayed protein and thetiter of eluted phage determined.

FIG. 3. Strategy for selecting internalizing antigen specific phageantibodies. Panel A, After two rounds of selection for internalizationon the basal breast cancer cell line MDAMB231, the pool of phageantibodies was first incubated with irrelevant control yeast to removeany yeast binding antibodies followed by panning on yeast displayingeither EphA2 ECD or CD44 domain 1. Panel B, The binding signal of thepolyclonal phage antibody pool to EphA2 ECD or CD44 domain 1 after 2rounds of panning was measured by using flow cytometry. The irrelevantcontrol yeast was stained with unselected phage antibody library (R0),round 1 (R1) and round 2 (R2) polyclonal phage. Panel C, Frequency ofantigen specific phage antibodies after one and two rounds of selectionon yeast displayed antigen. Binding frequency was determined byanalyzing 96 randomly picked phage antibodies for binding to yeastdisplayed antigen by flow cytometry. The induced yeast cells displayingan irrelevant protein, EphA2-ECD or CD44 domain 1 were stained withun-selected phage library (R0) and polyclonal phages from R1 and R2respectively. The un-induced yeast cells (yeast only), irrelevant phageantibody (phage control) and the un-selected phage antibody library wereused as control.

FIG. 4. Binding specificity of monoclonal phage antibodies from yeastantigen biopanning. Panel A, Binding of monoclonal phage antibodies toyeast displayed antigen domains as determined by flow cytometry. Theinduced yeast cells displaying an irrelevant protein (Y-CON), EphA2-ECD(Y-EphA2 ECD), CD44 link domain (Y-CD44 ECD D1) and CD44 full length ECD(Y-CD44 ECD) were stained with monoclonal phage antibodies isolated fromY-EphA2 and Y-CD44 D1 selections. Panel B shows the binding ofmonoclonal phage antibodies to MDAMB231 cells as determined by flowcytometry. Panel C shows the differential binding of monoclonal phageantibodies to breast cancer cell lines including luminal breast cancercell lines SUM52PE and MCF7, and basal breast cancer cell line MDAMB231.

FIG. 5. Characterization of scFv antibodies by western-blot and flowcytometry. Panel A, Anti-EphA2 scFv 2D6 or D2-1A7 and anti-CD44 scFvF2-1A6 were used to immunoprecipitate their target antigen from MDAMB231cells. Antigen was detected by Western blotting using either anti-EphA2antibody D7 (Upstate Biotech, Billerica) or anti-CD44 antibody Ab-4(NeoMarkers, Fremont). Panel B, EphA2 antibodies D2-1A7, D2-1A9, and 2D6compete with ephrin A1 for binding to MDAMB231 cells. The ability ofphage antibodies D2-1A7, D2-1A9, and 2D6 that bind to MDAMB231 cells inthe presence of increasing concentrations of the EphA2 ligand ephrin A1was determined by flow cytometry.

FIG. 6. Phage antibodies specific to EphA2 and CD44 are endocytosed byMDAMB231 cells. Cultured cells were incubated with irrelevant phage(panel A), anti-EphA2 phage D2-1A7 (panel B) and D2-1A9 (panel C),anti-CD44 phage F2-1A6 (panels D and E) for 3 hr at 37° C. followed byGlycine buffer wash. Endocytosis was determined by detection ofintracellular phage with anti-fd antibody, and analyzing by confocalmicroscopy.

FIG. 7. Amino acid sequences of full length V_(H) and V_(L) ofantibodies specific for CD44 from various clones. The framework andcomplementary determining regions are also indicated. SEQ ID NOs refersto the full length variable region.

FIG. 8. Amino acid sequences of full length V_(H) and V_(L) ofantibodies specific for EphA2 from various clones. The framework andcomplementary determining regions are also indicated. SEQ ID NOs refersto the full length variable region.

FIGS. 9A and 9B. Amino acid sequences and encoding nucleic acidsequences for full length antibodies from clone 2D6.

FIGS. 10A and 10B. Amino acid sequences and encoding nucleic acidsequences for full length scFv antibodies from clone D2-1A7.

FIGS. 11A and 11B. Amino acid sequences and encoding nucleic acidsequences for full length scFv antibodies from clone D2-1A9.

FIGS. 12A and 12B. Amino acid sequences and encoding nucleic acidsequences for full length scFv antibodies from clone D2-1B1.

FIGS. 13A and 13B. Amino acid sequences and encoding nucleic acidsequences for full length scFv antibodies from clone F2-1A6.

FIGS. 14A and 14B. Amino acid sequences and encoding nucleic acidsequences for full length scFv antibodies from clone F2-1H9.

FIGS. 15A and 15B. Amino acid (SEQ ID NO:19) and encoding nucleic acid(SEQ ID NO:20) sequences for full length scFv antibodies from cloneE8H11.

FIGS. 16A and 16B. Amino acid (SEQ ID NO:58) and encoding nucleic acid(SEQ ID NO:59) sequences for full length scFv antibodies from cloneE8H7.

FIGS. 17A and 17B. Amino acid (SEQ ID NO:60) and encoding nucleic acid(SEQ ID NO:61) sequences for full length scFv antibodies from cloneE8G12.

FIGS. 18A and 18B. Amino acid (SEQ ID NO:63) and encoding nucleic acid(SEQ ID NO:64) sequences for full length scFv antibodies from cloneE8F11.

FIGS. 19A and 19B. Amino acid (SEQ ID NO:72) and encoding nucleic acid(SEQ ID NO:73) sequences for full length scFv antibodies from cloneE8C9.

FIGS. 20A and 20B. Amino acid (SEQ ID NO:85) and encoding nucleic acid(SEQ ID NO:86) sequences for full length scFv antibodies from cloneD6G9.

FIGS. 21A and 21B. Amino acid (SEQ ID NO:127) and encoding nucleic acid(SEQ ID NO:128) sequences for full length scFv antibodies from cloneD6D3.

FIGS. 22A and 22B. Amino acid (SEQ ID NO:129) and encoding nucleic acid(SEQ ID NO:130) sequences for full length scFv antibodies from cloneA3H9.

FIGS. 23A and 23B. Amino acid (SEQ ID NO:138) and encoding nucleic acid(SEQ ID NO:139) sequences for full length scFv antibodies from cloneA3G3.

FIGS. 24A and 24B. Amino acid (SEQ ID NO:140) and encoding nucleic acid(SEQ ID NO:141) sequences for full length scFv antibodies from cloneA3D10.

FIGS. 25A and 25B. Amino acid (SEQ ID NO:310) and encoding nucleic acid(SEQ ID NO:311) sequences for full length scFv antibodies from cloneA3D1.

FIGS. 26A and 26B. Amino acid (SEQ ID NO:312) and encoding nucleic acid(SEQ ID NO:313) sequences for full length scFv antibodies from cloneA3C8.

FIGS. 27A and 27B. Amino acid (SEQ ID NO:314) and encoding nucleic acid(SEQ ID NO:315) sequences for full length scFv antibodies from clone1A3.

FIGS. 28A and 28B. Amino acid (SEQ ID NO:316) and encoding nucleic acid(SEQ ID NO:317) sequences for full length scFv antibodies from clone1A5.

FIGS. 29A and 29B. Amino acid (SEQ ID NO:318) and encoding nucleic acid(SEQ ID NO:319) sequences for full length scFv antibodies from clone1A8.

FIGS. 30A and 30B. Amino acid (SEQ ID NO:320) and encoding nucleic acid(SEQ ID NO:321) sequences for full length scFv antibodies from clone1A12.

FIGS. 31A and 31B. Amino acid (SEQ ID NO:322) and encoding nucleic acid(SEQ ID NO:323) sequences for full length scFv antibodies from clone1B2.

FIGS. 32A and 32B. Amino acid (SEQ ID NO:324) and encoding nucleic acid(SEQ ID NO:325) sequences for full length scFv antibodies from clone1C2.

FIGS. 33A and 33B. Amino acid (SEQ ID NO:326) and encoding nucleic acid(SEQ ID NO:327) sequences for full length scFv antibodies from clone1C7.

FIGS. 34A and 34B. Amino acid (SEQ ID NO:328) and encoding nucleic acid(SEQ ID NO:329) sequences for full length scFv antibodies from clone1D8.

FIGS. 35A and 35B. Amino acid (SEQ ID NO:330) and encoding nucleic acid(SEQ ID NO:331) sequences for full length scFv antibodies from clone15H11.

FIGS. 36A and 36B. Amino acid (SEQ ID NO:332) and encoding nucleic acid(SEQ ID NO:333) sequences for full length scFv antibodies from cloneD1C5.

FIGS. 37A and 37B. Amino acid (SEQ ID NO:334) and encoding nucleic acid(SEQ ID NO:335) sequences for full length scFv antibodies from cloneD1D1.

FIGS. 38A and 38B. Amino acid (SEQ ID NO:336) and encoding nucleic acid(SEQ ID NO:337) sequences for full length scFv antibodies from cloneH1B8 (also referred to herein as “HB8”).

FIGS. 39A and 39B. Amino acid (SEQ ID NO:338) and encoding nucleic acid(SEQ ID NO:339) sequences for full length scFv antibodies from cloneH1C2 (also referred to herein as “HC2”).

FIGS. 40A and 40B. Amino acid (SEQ ID NO:340) and encoding nucleic acid(SEQ ID NO:341) sequences for full length scFv antibodies from cloneH1C4 (also referred to herein as “HC4”).

FIGS. 41A and 41B. Amino acid (SEQ ID NO:342) and encoding nucleic acid(SEQ ID NO:343) sequences for full length scFv antibodies from cloneH1E3 (also referred to herein as “HE3”).

FIGS. 42A and 42B. Amino acid (SEQ ID NO:344) and encoding nucleic acid(SEQ ID NO:345) sequences for full length scFv antibodies from cloneH1F1 (also referred to herein as “HF1”).

FIGS. 43A and 43B. Amino acid (SEQ ID NO:346) and encoding nucleic acid(SEQ ID NO:347) sequences for full length scFv antibodies from cloneH1H3 (also referred to herein as “HH3”).

FIG. 44. Amino acid sequences of full length V_(H) and V_(L) ofantibodies specific for CD44 from various clones. The framework andcomplementary determining regions are also indicated. SEQ ID NOs referto the full length variable region. V_(H) sequences of E8H11 (SEQ IDNO:348); E8H7 (SEQ ID NO:349); E8G12 (SEQ ID NO:350); E8F11 (SEQ IDNO:351); E8C9 (SEQ ID NO:352); D6G9 (SEQ ID NO:353); and D6D3 (SEQ IDNO:354) are provided. V_(L) sequences of E8H11 (SEQ ID NO:355); E8H7(SEQ ID NO:356); E8G12 (SEQ ID NO:357); E8F11 (SEQ ID NO:358); E8C9 (SEQID NO:359); D6G9 (SEQ ID NO:360); and D6D3 (SEQ ID NO:361) are provided.

FIGS. 45A and 45B. Amino acid sequences of full length V_(H) and V_(L)of antibodies specific for CD44 from various clones. The framework andcomplementary determining regions are also indicated. SEQ ID NOs referto the full length variable region. V_(H) sequences of D1C5 (SEQ IDNO:362); D1D1 (SEQ ID NO:363); HB8 (SEQ ID NO:364); HC2 (SEQ ID NO:365);HC4 (SEQ ID NO:366); HE3 (SEQ ID NO:367); HF1 (SEQ ID NO:368); and HH3(SEQ ID NO:369) are provided. V_(L) sequences of D1C5 (SEQ ID NO:370);D1D1 (SEQ ID NO:371); HB8 (SEQ ID NO:372); HC2 (SEQ ID NO:373); HC4 (SEQID NO:374); HE3 (SEQ ID NO:375); HF1 (SEQ ID NO:376); and HH3 (SEQ IDNO:377) are provided.

FIG. 46. Amino acid sequences of full length V_(H) and V_(L) ofantibodies specific for EphA2 from various clones. The framework andcomplementary determining regions are also indicated. SEQ ID NOs referto the full length variable region. V_(H) sequences of A3H9 (SEQ IDNO:378); A3G3 (SEQ ID NO:379); A3D10 (SEQ ID NO:380); A3D1 (SEQ IDNO:381); and A3C8 (SEQ ID NO:382) are provided. V_(L) sequences of A3H9(SEQ ID NO:383); A3G3 (SEQ ID NO:384); A3D10 (SEQ ID NO:385); A3D1 (SEQID NO:386); and A3C8 (SEQ ID NO:387) are provided.

FIG. 47. Amino acid sequences of full length V_(H) and V_(L) ofantibodies specific for EphA2 from various clones. The framework andcomplementary determining regions are also indicated. SEQ ID NOs referto the full length variable region. V_(H) sequences of 1A3 (SEQ IDNO:388); 1A5 (SEQ ID NO:389); 1A8 (SEQ ID NO:390); 1A12 (SEQ ID NO:391);1B2 (SEQ ID NO:392); 1C2 (SEQ ID NO:393); 1C7 (SEQ ID NO:394); and 1D8(SEQ ID NO:395) are provided. V_(L) sequences of 1A3 (SEQ ID NO:396);1A5 (SEQ ID NO:397); 1A8 (SEQ ID NO:398); 1A12 (SEQ ID NO:399); 1B2 (SEQID NO:400); 1C2 (SEQ ID NO:401); 1C7 (SEQ ID NO:402); and 1D8 (SEQ IDNO:403) are provided.

DEFINITIONS

The following abbreviations may be used herein: CDR, complementaritydetermining region; Fab, antigen binding fragment of immunoglobulin withvariable domain and first constant domain; FACS, fluorescent activatedcell sorting; IgG, immunoglobulin G; K_(D), dissociation equilibriumconstant; k_(on), association rate constant; k_(off), dissociation rateconstant; mAb, monoclonal antibody; MFI, mean fluorescent intensity;PBS, phosphate buffered saline; PCR, polymerase chain reaction; scFv,single chain format of antibody variable regions; V_(H), heavy chainvariable region; V_(L), light chain variable region; TAA, tumorassociated antigen; EGFR, epidermal growth factor receptor; ECD,extracellular domain; IMAC, immobilized metal affinity chromatography;ILs, immunoliposomes; IPTG, Isopropyl-β-D-thiogalactopyranoside; 2-MEA,2-Mercaptoethylamine; DTT, Dithiothreitol; TEA, triethylamine; TBS-T,Tris-buffered saline Tween-20; Ni-NTA, Nickel-nitrilotriacetic acid; PE,phycoerythrin; HMEC, human mammary epithelial cell.

As used herein, “EphA2” refers to a member of the receptor tyrosinekinase family that can bind EphrinA ligands, and can also be named“epithelial cell kinase (ECK)”. The term “EphA2” can refer to anynaturally occurring isoforms of a EphA2. The amino acid sequence ofEphA2 is known and can be found as GenBank Accession No. NP_(—)004422.2.

As used herein, “CD44” refers to receptor for hyaluronic acid (HA), andcan also be called “phagocytic glycoprotein I”, “hyaluronate receptor”,or “CD44 antigen”. For example, the term “CD44” can refer to either anyone of naturally-occurring isoforms of a CD44. The amino acid sequenceof CD44 is known and the longest isoform of CD44 can be found as GenBankAccession No. NP_(—)000601.3.

The terms “polypeptide”, “peptide”, or “protein” are usedinterchangeably herein to designate a linear series of amino acidresidues connected one to the other by peptide bonds between thealpha-amino and carboxy groups of adjacent residues. In addition, theamino acids, in addition to the 20 “standard” genetically encodableamino acids, include amino acid analogs. Furthermore, it should be notedthat a dash at the beginning or end of an amino acid residue sequenceindicates either a peptide bond to a further sequence of one or moreamino acid residues or a covalent bond to a carboxyl or hydroxyl endgroup. However, the absence of a dash should not be taken to mean thatsuch peptide bonds or covalent bond to a carboxyl or hydroxyl end groupis not present, as it is conventional in representation of amino acidsequences to omit such.

“Antibody” encompasses compositions comprising an antigen-bindingprotein, individually or as a preparation comprising a pluralitythereof, having one or more polypeptides that can be geneticallyencodable by immunoglobulin genes, or fragments of immunoglobulin genes,or that comprise CDRs obtainted or derived from immunoglobulins, andwhich bind an antigen of interest. Light chains are classified as eitherkappa or lambda. Heavy chains can be classified as gamma, mu, alpha,delta, or epsilon, which in turn define the immunoglobulin classes, IgG,IgM, IgA, IgD and IgE, respectively.

An example of an antibody is one having a structural unit of a tetramercomposed of two pairs of polypeptide chains, each pair having one“light” and one “heavy” chain. The N-terminal portion of each chaindefines a variable region that mediates antigen binding. The termsvariable light chain (V_(L)) and variable heavy chain (V_(H)) refer tolight and heavy chains respectively.

“Antibody” also encompasses single-chain antibodies that contain a heavychain and a light chain linked together as a single polypeptide, each ofsuch linked heavy or light chains nonetheless being referred to hereinas a heavy chain or a light chain. Antibody may also refer to heavychain-only antibodies such as heavy chain antibodies or HCAbs.

As noted above, “antibody” encompasses intact immunoglobulins as wellantigen-binding fragments of antibodies. Thus, the term “antibody”, asused herein also includes an antigen-binding portion of an antibody,which can be produced by the modification of whole antibodies orsynthesized de novo using recombinant DNA methodologies. Examplesinclude, but are not limited to, Fab′, Fab′₂, scFv, nanobodies,unibodies, and diabodies. A “nanobody” refers to the smallestantigen-binding fragment of a single chain antibody, also referred to asa V_(H)H or single-domain antibodies (dAbs).

“Monoclonal antibody” refers to a composition comprising one or moreantibodies obtained from a population of substantially homogeneousantibodies, i.e., a population the individual antibodies of which areidentical except for any naturally occurring mutations that may bepresent in minor amounts. Monoclonal antibodies are highly specific,being directed against a single antigenic site and generally to a singleepitope on an antigen. The modifier “monoclonal” indicates the characterof the antibody as being obtained from a substantially homogeneouspopulation of antibodies, and does not require that the antibody beproduced by any particular method or be the only antibody in thecomposition.

A single chain Fv (“scFv”) polypeptide is a covalently linkedV_(H)::V_(L) heterodimer which may be expressed from a nucleic acidincluding V_(H)- and V_(L)-encoding sequences either joined directly orjoined by a peptide-encoding linker (Huston, et al. (1988) Proc. Nat.Acad. Sci. USA, 85: 5879-5883). A number of structures are available forconverting the light and heavy polypeptide chains from an antibody Vregion into a scFv molecule which will fold into a three dimensionalstructure substantially similar to the structure of an antigen-bindingsite. In addition to being diabodies, the scFvs can also be present astribodies or tetrabodies.

It should be noted that while various antibody fragments are defined interms of the digestion of an intact antibody, one of skill willappreciate that such fragments may be synthesized de novo eitherchemically or by utilizing recombinant DNA methodology.

The term “antibody” encompasses polyclonal and monoclonal antibodies,and further encompasses antibodies of any class (e.g., IgM, IgG, andsubclasses thereof). “Antibody” also encompasses hybrid antibodies,bispecific antibodies, heteroantibodies, chimeric antibodies, humanizedantibodies, and functional fragments thereof which retain antigenbinding. “Bispecific antibodies” may resemble single antibodies (orantibody fragments) but have two different antigen binding sites(variable regions). Heteroantibodies refers to two or more antibodies,or antibody binding fragments (e.g., Fab) linked together, each antibodyor fragment having a different specificity. The antibodies may beconjugated to other moieties, and/or may be bound to a support (e.g., asolid support), such as a polystyrene plate or bead, test strip, and thelike.

An immunoglobulin light or heavy chain variable region is composed of a“framework” region (FR) interrupted by three hypervariable regions, alsocalled “complementarity determining regions” or “CDRs”. The extent ofthe framework region and CDRs can be defined based on databases known inthe art. See, for example, “Sequences of Proteins of ImmunologicalInterest,” E. Kabat et al., Sequences of proteins of immunologicalinterest, 4th ed. U.S. Dept. Health and Human Services, Public HealthServices, Bethesda, Md. (1987), Lefranc et al. IMGT, the internationalImMunoGeneTics information System®. Nucl. Acids Res., 2005, 33:D593-D597(www.imgt.org/textes/IMGTScientificChart/), and/or V Base atvbase.mrc-cpe.cam.ac.uk/). The sequences of the framework regions ofdifferent light or heavy chains are relatively conserved within aspecies. The framework region of an antibody, that is the combinedframework regions of the constituent light and heavy chains, serves toposition and align the CDRs. The CDRs are primarily responsible forbinding to an epitope of an antigen. All CDRs and framework provided bythe present disclosure are defined according to Kabat et al., supra,unless otherwise indicated.

An “anti-EphA2 antibody” or “anti-CD44 antibody” refers to an antibodythat, specifically binds to EphA2 or CD44, preferably with highaffinity. A specific antibody for EphA2 or CD44 does not exhibitcomparable binding to other antigens unrelated to EphA2 or CD44 relativeto the binding of EphA2 or CD44.

The term “high affinity” when used with respect to an antibody refers toan antibody that specifically binds to (“recognizes”) its target(s) withan affinity (K_(D)) value less than or equal to 10⁻⁶ M, less than 10⁻⁷M, less than 10⁻⁸ M, preferably less than 10⁻⁹ M, less than 10⁻¹⁰ M, orless than 10⁻¹¹ M. A lower K_(D) value corresponds to a higher bindingaffinity (i.e., stronger binding) so that a K_(D) value of 10⁻⁷indicates a higher binding affinity than a K_(D) value of 10⁻⁶.

An “antigen-binding site” or “binding portion” refers to a part of anantibody molecule (e.g. fragment of an immunoglobulin molecule or scFv)that participates in immunoreactive antigen binding. The antigen bindingsite is formed by amino acid residues of the N-terminal variable (“V”)regions of the heavy (“H”) and/or light (“L”) chains. Three highlydivergent stretches within the V regions of the heavy and light chainsare referred to as “hypervariable regions” which are interposed betweenmore conserved flanking stretches known as “framework regions” or “FRs”.Thus, the term “FR” refers to amino acid sequences that are naturallyfound between and adjacent to hypervariable regions in immunoglobulins.In a tetrameric antibody molecule, the three hypervariable regions of alight chain and the three hypervariable regions of a heavy chain aredisposed relative to each other in three dimensional space to form anantigen binding “surface”. This surface mediates recognition and bindingof the target antigen. The three hypervariable regions of each of theheavy and/or light chains are referred to as “complementaritydetermining regions” or “CDRs” and are determined, for example based onKabat et al., supra.

A “F2-1A6 antibody” refers to an antibody expressed by clone F2-1A6 orto an antibody synthesized in other manners, but having the same CDRsand optionally, the same framework regions as the antibody expressed byclone F2-1A6, and having substantially the same antigen bindingspecificity. Similarly, antibodies 2D6, D2-1A7, D2-1A9, D2-1B1, F2-1H9,and the like (e.g., antibodies E8H11, E8H7, E8G12, E8F11, and E8C9;antibodies D6G9 and D6D3; antibodies D1C5 and D1D1; antibodies HB8, HC2,HC4, HE3, HF1, and HH3; antibodies A3H9, A3G3, A3D10, A3D1, and A3C8;antibodies 1A3, 1A5, 1A8, 1A12, 1B2, 1C2, 1C7, and 1D8; and antibody15H11) refer to antibodies expressed by the corresponding clone(s)and/or to antibodies synthesized in other manners, but having the sameCDRs and optionally, the same framework regions as the referencedantibodies, and having substantially the same antigen bindingspecificity. The CDRs of these antibodies are defined by Kabat et al.,supra, as shown in FIGS. 7, 8, and 44-47, and tables below.

An “epitope” is a site on an antigen (e.g. a site on the EphA2extracellular domain (ECD)) to which an antibody binds. Epitopes can beformed both from contiguous amino acids or noncontiguous amino acidsjuxtaposed by folding (e.g., tertiary folding) of a protein. Epitopesformed from contiguous amino acids are typically retained on exposure todenaturing solvents whereas epitopes formed by folding are typicallylost on treatment with denaturing solvents. An epitope typicallyincludes at least 3, and more usually, at least 5 or 8-10 amino acids ina linear or spatial conformation. Methods of determining spatialconformation of epitopes include, for example, x-ray crystallography and2-dimensional nuclear magnetic resonance. See, e.g., Epitope MappingProtocols in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed(1996). Several commercial laboratories offer epitope mapping services.Epitopes bound by an antibody immunoreactive with a membrane associatedantigen can reside on the surface of the cell (e.g. in the extracellularregion of a transmembrane protein), so that such epitopes are consideredcell-surface accessible, solvent accessible, and/or cell-surfaceexposed.

“Isolated” refers to an entity of interest (e.g., a protein, e.g., anantibody) that is in an environment different from that in which theentity may naturally occur. An “isolated” entity is separated from allor some of the components that accompany it in nature and may besubstantially enriched. “Isolated” also refers to the state of an entityseparated from all or some of the components that accompany it duringmanufacture (e.g., chemical synthesis, recombinant expression, culturemedium, and the like). A subject antibody can be substantially pure.“Substantially pure” can refer to compositions in which at least 75%, atleast 85%, at least 90%, at least 95%, at least 98%, at least 99%, ormore than 99%, of the total composition is the entity of interest (e.g.,a subject antibody).

The phrases “specifically bind(s) to,” “specific for,” “immunoreactive”and “immunoreactivity,” and “antigen binding specificity,” whenreferring to an antibody, refer to a binding reaction with an antigenwhich is highly preferential to the antigen or a fragment thereof, so asto be determinative of the presence of the antigen in the presence of aheterogeneous population of antigens (e.g., proteins and otherbiologics, e.g., in a tissue). Thus, under designated immunoassayconditions, the specified antibodies bind to a particular antigen and donot bind in a significant amount to other antigens present in thesample. Specific binding to an antigen under such conditions may requirean antibody that is selected for its specificity for a particularantigen. For example, anti-CD44 antibodies can specifically bind toCD44, and do not exhibit comparable binding (e.g., do not exhibitdetectable binding) to other proteins present in a tissue sample. SeeHarlow and Lane (1988) Antibodies, A Laboratory Manual, Cold SpringHarbor Publications, New York, for a description of immunoassay formatsand conditions that can be used to determine specific immunoreactivity.

DETAILED DESCRIPTION

Antibodies that specifically bind to tumor associated antigen CD44 andantibodies that specifically bind to tumor associated antigen EphA2 aredisclosed herein, as well as related compositions and methods of usethereof. Methods of use encompass cancer therapies, diagnostics, andscreening methods.

Where the antibodies are specific for CD44, the antibodies contain atleast one, two, or all three CDRs of the V_(H) of the antibody from(e.g., obtained by the expression of) clone F2-1A6, F2-1H9, E8H11, E8H7,E8G12, E8F11, E8C9, D6G9, D6D3, D1C5, D1D1, HB8, HC2, HC4, HE3, HF1, orHH3. The antibodies also encompass those containing at least one, two,or all three CDRs of the V_(L) of the antibody from clone F2-1A6,F2-1H9, E8H11, E8H7, E8G12, E8F11, E8C9, D6G9, D6D3, D1C5, D1D1, HB8,HC2, HC4, HE3, HF1, or HH3. The antibodies also encompass thosecontaining at least one, two, or all three CDRs independently selectedfrom each of the V_(L) and the V_(H) of an antibody from clone F2-1A6,F2-1H9, E8H11, E8H7, E8G12, E8F11, E8C9, D6G9, D6D3, D1C5, D1D1, HB8,HC2, HC4, HE3, HF1, or HH3. Alternately the antibodies compete forbinding to CD44 with (e.g., bind to the same epitope as) and antibodyfrom clone F2-1A6, F2-1H9, E8H11, E8H7, E8G12, E8F11, E8C9, D6G9, D6D3,D1C5, D1D1, HB8, HC2, HC4, HE3, HF1, or HH3.

Where the antibodies are specific for EphA2, the antibodies contain atleast one, two or all three heavy chain (V_(H)) complementaritydetermining region(s) (CDR(s)) of an antibody from clone 2D6, D2-1A7,D2-1A9, D2-1B1, A3H9, A3G3, A3D10, A3D1, A3C8, 1A3, 1A5, 1A8, 1A12, 1B2,1C2, 1C7, 1D8, or 15H11. The antibodies also encompass those containingat least one, two or all three light chain (V_(L)) complementaritydetermining region(s) (CDR(s)) of an antibody from clone 2D6, D2-1A7,D2-1A9, D2-1B1, A3H9, A3G3, A3D10, A3D1, A3C8, 1A3, 1A5, 1A8, 1A12, 1B2,1C2, 1C7, 1D8, or 15H11. The antibodies also encompass those containingat least one, two, or all three CDRs independently selected from each ofthe V_(L) and the V_(H) of an antibody from clone 2D6, D2-1A7, D2-1A9,D2-1B1, A3H9, A3G3, A3D10, A3D1, A3C8, 1A3, 1A5, 1A8, 1A12, 1B2, 1C2,1C7, 1D8, or 15H11. Alternately the antibodies compete for binding toEphA2 with (e.g., bind to the same epitope as) and antibody from clone2D6, D2-1A7, D2-1A9, D2-1B1, A3H9, A3G3, A3D10, A3D1, A3C8, 1A3, 1A5,1A8, 1A12, 1B2, 1C2, 1C7, 1D8, or 15H11.

An antibody of the present disclosure may also contain all V_(H) CDRsand/or V_(L) CDRs of an antibody from clone F2-1A6, F2-1H9, 2D6, D2-1A7,D2-1A9, D2-1B1, E8H11, E8H7, E8G12, E8F11, E8C9, D6G9, D6D3, D1C5, D1D1,HB8, HC2, HC4, HE3, HF1, HH3, A3H9, A3G3, A3D10, A3D1, A3C8, 1A3, 1A5,1A8, 1A12, 1B2, 1C2, 1C7, 1D8, or 15H11. The antibodies may containfull-length V_(H) chains of an antibody from clone F2-1A6, F2-1H9, 2D6,D2-1A7, D2-1A9, D2-1B1, E8H11, E8H7, E8G12, E8F11, E8C9, D6G9, D6D3,D1C5, D1D1, HB8, HC2, HC4, HE3, HF1, HH3, A3H9, A3G3, A3D10, A3D1, A3C8,1A3, 1A5, 1A8, 1A12, 1B2, 1C2, 1C7, 1D8, or 15H11. The antibodies canalso or alternatively contain full-length V_(L) chains of an antibodyfrom clone F2-1A6, F2-1H9, 2D6, D2-1A7, D2-1A9, D2-1B1, E8H11, E8H7,E8G12, E8F11, E8C9, D6G9, D6D3, D1C5, D1D1, HB8, HC2, HC4, HE3, HF1,HH3, A3H9, A3G3, A3D10, A3D1, A3C8, 1A3, 1A5, 1A8, 1A12, 1B2, 1C2, 1C7,1D8, or 15H11.

The antibody may be a full length antibody (i.e., an antibody comprisingat least one full-length V_(H) sequence and at least one full lengthV_(L) sequence) or a fragment such as a single chain Fv (scFv), a Fab, a(Fab′)₂, an (ScFv)₂, and the like. The antibody may be an IgG (e.g.,IgG₂) or any other isotype, or may be a bispecific antibody.

The antibodies may be conjugated, such as to an anti-cancer drug, alabel, or to a moiety that improves or promotes serum half-life (e.g.poly(ethylene glycol) (PEG)), endocytosis, or another biologicalfunction or characteristic. The antibody may also be in a compositioncomprising a pharmaceutically acceptable excipient, e.g., a compositionsuitable for injection (e.g., in a unit dosage formulation). The presentdisclosure also provides compositions that include one or more differentantibodies selected from the antibodies described herein and/orantibodies comprising one or more CDRs from these antibodies, and/or oneor more antibodies comprising mutants or derivatives of theseantibodies. The composition may include one or more antibodies, such asF2-1A6, F2-1H9, E8H11, E8H7, E8G12, E8F11, E8C9, D6G9, D6D3, D1C5, D1D1,HB8, HC2, HC4, HE3, HF1, HH3, 2D6, D2-1A7, D2-1A9, D2-1B1, A3H9, A3G3,A3D10, A3D1, A3C8, 1A3, 1A5, 1A8, 1A12, 1B2, 1C2, 1C7, 1D8, or 15H11.

Methods of the present disclosure include those that provide foradministering one or more subject antibodies as disclosed herein in anamount effective to treat a subject having cancer expressing theantigen(s) specifically bound by the subject antibody or antibodies. Theantibodies provided by this disclosure can also be used fordiagnosis/prognosis or imaging of cancer.

Nucleic acids provided herein encode one or more antibodies that aredescribed herein. Host cells containing such nucleic acids are alsoprovided herein, as well as those that produce the subject antibodies(e.g. by secretion). Kits are also provided for preparing compositionscontaining the subject antibodies or for carrying out the subjectmethods.

Antibodies

Preferred antibodies have a high affinity (e.g., exhibit K_(D) values of10⁻⁷ M or lower) to one or more of the tumor associated antigens (TAA),EphA2 or CD44, that are cell-surface exposed on cancer cells during atleast some portion of the cell cycle. Antibodies having lower affinity(e.g., having K_(D) values of from 10⁻⁵ M to 10⁻⁶ M) for EphA2 or CD44are also contemplated. Cancer cells, for example, include those derivedfrom breast cancer cells (e.g. MDAMB231) and others. The subjectantibodies include those that are internalized into the cell uponbinding to antigen, e.g., an antigen on the surface of a livingmammalian cell, e.g. by endocytosis, such as receptor-mediatedendocytosis.

The subject antibodies include those that competitively bind to anepitope of CD44 with an antibody from clone F2-1A6, F2-1H9, E8H11, E8H7,E8G12, E8F11, E8C9, D6G9, D6D3, D1C5, D1D1, HB8, HC2, HC4, HE3, HF1, orHH3. The antibodies also encompass those that competitively bind to anepitope on EphA2 with an antibody from clone 2D6, D2-1A7, D2-1A9,D2-1B1, A3H9, A3G3, A3D10, A3D1, A3C8, 1A3, 1A5, 1A8, 1A12, 1B2, 1C2,1C7, 1D8, or 15H11. The ability of a particular antibody to recognizethe same epitope as another antibody can be determined by the ability ofone antibody to competitively inhibit binding of the second antibody(e.g., competitively bind) to the antigen (e.g., as determined bycompetitive binding assays such as those disclosed in US patentpublication No. 20090291085). Competitive inhibition of binding may alsobe referred to as cross-reactivity of antibodies.

Any of a number of competitive binding assays can be used to measurecompetition between two antibodies to the same antigen. For example, asandwich ELISA assay can be used for this purpose. Means of assaying forcross-reactivity are well known to those of skill in the art (see, e.g.,Dowbenko et al. (1988) J. Virol. 62: 4703-4711).

An antibody is considered to competitively inhibit binding of a secondantibody to an antigen if binding of the second antibody to the antigenis reduced by at least 30%, usually at least about 40%, 50%, 60% or 75%,and often by at least about 90%, in the presence of the first antibodyusing any of the assays used to assess competitive binding.

This can be ascertained by providing one or more isolated target TAA(s),EphA2 and/or CD44, attached to a solid support and assaying the abilityof an antibody to bind to the target TAA or to compete with an antibodydescribed herein for binding to the target TAA (e.g. using surfaceplasmon resonance).

As noted above, the subject antibodies encompass those that compete withone or more of the following antibodies: 2D6, D2-1A7, D2-1A9, D2-1B1,F2-1A6, F2-1H9, E8H11, E8H7, E8G12, E8F11, E8C9, D6G9, D6D3, D1C5, D1D1,HB8, HC2, HC4, HE3, HF1, HH3, A3H9, A3G3, A3D10, A3D1, A3C8, 1A3, 1A5,1A8, 1A12, 1B2, 1C2, 1C7, 1D8, and 15H11. In addition, the antibodiescan have a binding affinity for EphA2 or CD44 comparable to or greaterthan about 1×10⁻⁶ M (i.e., the antibodies can exhibit K_(D) values lowerthan 10⁻⁶ M, e.g., about 10⁻⁷ M, 10⁻⁸ M, 10⁻⁹ M, 10⁻¹⁰ M or an evenhigher binding affinity such as a K_(D) value of about 10⁻¹¹ M or 10⁻¹²M).

In a related embodiment, the subject antibodies encompass those thatbind to the same epitope as F2-1A6 or F2-1H9 or to the epitope of 2D6,D2-1A7, D2-1A9, or D2-1B1, or to the epitope of E8H11, E8H7, E8G12,E8F11, E8C9, D6G9, D6D3, D1C5, D1D1, HB8, HC2, HC4, HE3, HF1, or HH3, orto the epitope of A3H9, A3G3, A3D10, A3D1, A3C8, 1A3, 1A5, 1A8, 1A12,1B2, 1C2, 1C7, 1D8, or 15H11. Epitope mapping can be performed usingpairs of antibodies at concentrations resulting in near saturation andat least 100 relative units (RU) of antibody bound. The amount ofantibody bound is determined for each member of a pair, and then the twoantibodies are mixed together to give a final concentration equal to theconcentration used for measurements of the individual antibodies.Antibodies recognizing different epitopes show an essentially additiveincrease in the RU bound when injected together, while antibodiesrecognizing identical epitopes show only a minimal increase in RU.Antibodies may be said to be cross-reactive if, when “injected” togetherthey show an essentially additive increase (e.g. an increase by at leasta factor of about 1.4, an increase by at least a factor of about 1.6, oran increase by at least a factor of about 1.8 or 2.)

Epitopes of antibodies can also be ascertained by a number of otherstandard techniques (see, e.g., Geysen et al (1987) J. Immunol. Meth102:259-274). This technique involves the synthesis of large numbers ofoverlapping peptides of EphA2 or CD44. The synthesized peptides are thenscreened against one or more of the prototypical antibodies (e.g., 2D6,etc.) and the characteristic epitopes specifically bound by theseantibodies can be identified by binding specificity and affinity. Theepitopes thus identified can be conveniently used for competitive assaysas described herein to identify cross-reacting antibodies.

The peptides for epitope mapping can be conveniently prepared using“Multipin” peptide synthesis techniques (see, e.g., Geysen et al (1987)Science 235:1184-1190). Using the known sequence of one or more EphA2and/or CD44, overlapping polypeptide sequences can be synthesizedindividually in a sequential manner on plastic pins in an array of oneor more 96-well microtest plate(s).

Anti-CD44

Antibodies of the present disclosure include those that specificallybind CD44. Anti-CD44 antibodies encompass those that competitively bindto an epitope (e.g. in domain 1) of CD44 with F2-1A6, F2-1H9, E8H11,E8H7, E8G12, E8F11, E8C9, D6G9, D6D3, D1C5, D1D1, HB8, HC2, HC4, HE3,HF1, or HH3. The epitope bound by anti-CD44 antibodies reside in acontiguous amino acid sequence of CD44 from residue position 21-169, asset forth below.

(SEQ ID NO: 1) QIDLNITCRFAGVFHVEKNGRYSISRTEAADLCKAFNSTLPTMAQMEKALSIGFETCRYGFIEGHVVIPRIHPNSICAANNTGVYILTSNTSQYDTYCFNASAPPEEDCTSVTDLPNAFDGPITITIVNRDGTRYVQKGEYRTN PEDIY

The residue position numbers of CD44 are determined based on thesequence set forth in GenBank Accession No. NP_(—)000601.3 or UniProtAccession No. P16070.

Antigens that share similar epitopes as CD44 can also be binding targetsof subject antibodies. When bound to CD44, a subject antibody can beinternalized by the cell expressing the CD44 protein.

Epitopes for which anti-CD44 antibodies have affinity are cell-surfaceexposed and solvent-accessible on many cancer cells, particularly on theplasma membrane of cells. The epitopes can be accessible to the subjectantibodies when the cells are live. For example, the epitopes may bepresent on cancer cells derived from breast cancers, colon cancers,adenoma, head and neck squamous cell carcinoma (HNSCC), prostatecancers, pancreatic cancers, etc. Cancers cells for which anti-CD44antibodies have affinity may also be from any cancer that is metasticand/or has metastatic potential.

Additional examples of subject antibodies encompass those that have thesame binding specificities and comprise at least two CDRs that eachindependently shares at least about 80%, at least about 87%, at leastabout 93%, at least about 94%, or up to 100% amino acid sequenceidentity with the amino acid sequence of a V_(H) CDR of antibodies shownin FIGS. 7, 44, and 45 (45A and 45B) and in tables below (e.g. V_(H)CDR1 of F2-1A6, F2-1H9, E8H11, E8H7, E8G12, E8F11, E8C9, D6G9, D6D3,D1C5, D1D1, HB8, HC2, HC4, HE3, HF1, or HH3). The subject antibody canalso include all three CDRs from any V_(H) CDRs of each antibody shownin FIGS. 7, 44, and 45, such that each V_(H) CDR in the subject antibodyis selected from a single antibody shown in FIGS. 7, 44, and 45, ortables below (e.g., Table 1) and each V_(H) CDR independently shares atleast about 80%, at least about 87%, at least about 93%, at least about94%, or up to 100% amino acid sequence identity with the amino acidsequence of the V_(H) CDR of the antibody shown in FIG. 7, 44, or 45 ortables below (e.g., Table 1). For example, the heavy chain of a subjectantibody can contain two V_(H) CDRs or all three V_(H) CDRs of F2-1A6.Alternatively, the heavy chain can contain two V_(H) CDRs or all threeV_(H) CDRs of F2-1H9. As further examples, the heavy chain of a subjectantibody can contain two V_(H) CDRs or all three V_(H) CDRs of any oneof E8H11, E8H7, E8G12, E8F11, E8C9, D6G9, D6D3, D1C5, D1D1, HB8, HC2,HC4, HE3, HF1, and HH3.

Similarly for the light chain, a subject antibody will have the samebinding specificity and can contain at least two CDRs that are eachindependently at least about 80%, at least about 87%, at least about93%, at least about 94%, or up to 100% amino acid sequence identity withthe amino acid sequence of a V_(L) CDR of each antibody shown in FIGS.7, 44, and 45 (45A and 45B) or tables below (e.g. V_(L) CDR1 of F2-1A6;e.g., V_(L) CDR1 of E8H11, E8H7, E8G12, E8F11, E8C9, D6G9, D6D3, D1C5,D1D1, HB8, HC2, HC4, HE3, HF1, or HH3). The subject antibody can also oralternatively include all three V_(L) CDRs from any of the antibodiesshown in FIGS. 7, 44, and 45, or tables below (e.g., Table 2) and eachV_(L) CDR independently shares at least about 80%, at least about 87%,at least about 93%, at least about 94%, or up to 100% amino acidsequence identity with the amino acid sequence of the V_(L) CDR of theantibody shown in FIG. 7, 44, or 45, or tables below. For example, thelight chain of a subject antibody can contain two V_(L) CDRs or allthree V_(L) CDRs of F2-1A6. Alternatively, the light chain can containtwo V_(L) CDRs or all three V_(L) CDRs of F2-1H9. As further examples,the light chain of a subject antibody can contain two V_(L) CDRs or allthree V_(L) CDRs of E8H11, E8H7, E8G12, E8F11, E8C9, D6G9, D6D3, D1C5,D1D1, HB8, HC2, HC4, HE3, HF1, or HH3.

Optionally, antibodies can contain the same (i.e. 100% identity),similar, or different framework sequences (FR) in any of correspondingframework sequences in the heavy or light chain provided in FIGS. 7, 44,and 45 or tables below (e.g., Tables 1 and 2, below), so long as bindingspecificity is substantially maintained. Where the framework sequencesare similar, the framework may be at least about 85%, at least about86%, at least about 90%, at least about 93%, at least about 96%, atleast about 98%, or up to 100% identity to a corresponding frameworksequence in any of antibodies shown in FIGS. 7, 44, and 45 or tablesbelow (e.g., Tables 1 and 2, below).

An antibody of the present disclosure may therefore contain afull-length V_(H) and/or full length V_(L) sequence that is at least 80%identity, at least 85%, at least 90%, at least 95%, up to 100% aminoacid sequence identity to a full-length V_(H) or V_(L) sequence shown inFIG. 7, 44, or 45 or tables below. For example, a subject antibody cancontain the full length V_(H) and/or full length V_(L) of F2-1A6.Alternatively, the subject antibody can contain the full length V_(H)and/or full length V_(L) of F2-1H9. As further examples, a subjectantibody can contain the full length V_(H) and/or full length V_(L) ofE8H11, E8H7, E8G12, E8F11, E8C9, D6G9, D6D3, D1C5, D1D1, HB8, HC2, HC4,HE3, HF1, or HH3. Examples of antibodies of the present disclosure thatbind to CD44 are listed in tables below (e.g., Tables 1 and 2).

TABLE 1Heavy chain FRs and CDRs of antibodies that specifically bind CD44 cloneV_(H) FR1 V_(H) CDR1 V_(H) FR2 F2-1A6 QVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIG WVRQMPGKGLEWMG (SEQ ID NO: 2) (SEQ ID NO: 3) (SEQ ID NO: 4) F2-1H9QVQLQESGGGLVQPGGSLRLSCAASGFTFS SYRMH WVRQAPGKGLEWVA (SEQ ID NO: 5)(SEQ ID NO: 6) (SEQ ID NO: 7) D1C5 QVQLQQSGGGVVQPGRSLRLSCAASGFTFS SYGIHWVRQAPGKGLEWVA (SEQ ID NO: 34) (SEQ ID NO: 36) SEQ ID NO: 7) D1D1QVQLVESGGGLVKPGGSLRLSCAASGFTFS TYTMS WARQAPGKGLEWVS (SEQ ID NO: 24)(SEQ ID NO: 31) (SEQ ID NO: 39) HB8 QVQLVESGGGVVQPGRSLRLSCAASGFTFS NYAMIWVRQAPGKGLEWVS (SEQ ID NO: 131) (SEQ ID NO: 132) (SEQ ID NO: 133) HC2QVQRVESGGGVVQPGRSLRLSCAASGFTFS SYAMS WVRQAPGKGLEWVS (SEQ ID NO: 134)(SEQ ID NO: 32) (SEQ ID NO: 133) HC4 QVQLVESGGGLVQPGGSLRLSCAASGFTFSTLAMG WVRQAPGKGLEWVS (SEQ ID NO: 135) (SEQ ID NO: 42) (SEQ ID NO: 133)HE3 QVQLQESGGGLVKPGGSLRLSCEASGFTFS SYSMN WVRQAPGKGLEWVS (SEQ ID NO: 497)(SEQ ID NO: 52) (SEQ ID NO: 133) HF1 QVQLVESGGGLVQPGGSLRLSCAASGFTFSNYALI WVRQAPGKGLEWVS (SEQ ID NO: 74) (SEQ ID NO: 78) (SEQ ID NO: 133)HH3 QVQLQESGGGLVQPGGSLRLSCAASGFTFS SYAMS WVRQAPGKGLEWVS (SEQ ID NO: 5)(SEQ ID NO: 32) (SEQ ID NO: 133) E8H11 QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMH WVRQAPGKGLEWVA (SEQ ID NO: 131) (SEQ ID NO: 41) (SEQ ID NO: 7)E8H7 QVQLQESGGGLVQPGGSLRLSCSASGFTFS SYAMH WVRQAPGKGLEYVS(SEQ ID NO: 183) (SEQ ID NO: 35) (SEQ ID NO: 184) E8G12QVQLVQSGGGLVQPGGSLRLSCAASGFTFS SYAMS WVRQAPGKGLEWVS (SEQ ID NO: 185)(SEQ ID NO: 32) (SEQ ID NO: 133) E8F11 QVQLQQSGGGLVQPGGSLRLSCAASGFTFSSYAMS WVRQAPVKGLEWVS (SEQ ID NO: 186) (SEQ ID NO: 32) (SEQ ID NO: 187)E8C9 QVNLRESGGGLVKPGGSLRLSCAASGFTFS SYAMS WVRQAPGKGLEWVS(SEQ ID NO: 188) (SEQ ID NO: 32) (SEQ ID NO: 133) D6G9QVQLVESGGGLVQPGGSLRLSCAASGFTFS SYAMG WVRQAPGKGLEWVS (SEQ ID NO: 135)(SEQ ID NO: 189) (SEQ ID NO: 133) D6D3 QVQLQESGGGLVQPGGSLRLSCAASGFTFSSYAM WVRQAPGKGLEWVS (SEQ ID NO: 5) (SEQ ID NO: 32) (SEQ ID NO: 133)clone V_(H) CDR2 V_(H) FR3 F2-1A6 IIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCAR (SEQ ID NO: 8) (SEQ ID NO: 9) F2-1H9AVKQDGSEKYYLDSVKG RFTISRDNAKSSLYLQMDSLSVEDTAVYYCAR (SEQ ID NO: 10)(SEQ ID NO: 11) D1C5 VISYDGSNKYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK(SEQ ID NO: 45) (SEQ ID NO: 50) D1D1 AISADGAGTYYGDSVKGRFTVSRDNFKSTLYLQMNRLRAEDTAVYYCAK (SEQ ID NO: 82) (SEQ ID NO: 142) HB8AITGGGGSTYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTALYYCAK (SEQ ID NO: 143)(SEQ ID NO: 144) HC2 AISGSGGSTYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK(SEQ ID NO: 145) (SEQ ID NO: 50) HC4 AISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 145) (SEQ ID NO: 46) HE3AISGSGGSTYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK (SEQ ID NO: 145)(SEQ ID NO: 50) HF1 AISGSGSGTYYADSVKG RFTISRDNSKNTLYLQMNTLRAEDTALYYCAK(SEQ ID NO: 146) (SEQ ID NO: 147) HH3 AITSSGGRTYYADSVRGRLTISRDNSKNTLYLQMNTLRAEDTAVYYCAK (SEQ ID NO: 148) (SEQ ID NO: 149) E8H11VISYDGSNKYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK (SEQ ID NO: 45)(SEQ ID NO: 50) E8H7 AISSNGGSTYYADSVKG RFTISRDNSKNTLYLQMSSLRAEDTAVYYCVA(SEQ ID NO: 190) (SEQ ID NO: 191) H8G12 AISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK (SEQ ID NO: 145) (SEQ ID NO: 50) E8F11AISGSGGSTYYADSVKG RFTISRDNSKNTLYLQMNSLRIEDTAVYYCGR (SEQ ID NO: 145)(SEQ ID NO: 192) E8C9 VIS-DGSTTYYADSVKG RFTISRDNSKNMLYLQTNSLRAEDTAVYYCAK(SEQ ID NO: 193) (SEQ ID NO: 194) D6C9 AISASGGSTYYADSVKGRFTISRDNSKNTLYLQLNSLRAEDTAVYYCAK (SEQ ID NO: 195) (SEQ ID NO: 196) D6D3AITSSGGRTYYADSVRG RLTISRDNSKNTLYLQMNTLRAEDTAVYYCAK (SEQ ID NO: 148)(SEQ ID NO: 149) clone V_(H) CDR3 V_(H) FR4 F2-1A6 RLHGPFYFDYWGQGTLVTVSS (SEQ ID NO: 12) (SEQ ID NO: 13) F2-1H9 GLRT MGQGTLVTVSS(SEQ ID NO: 14) (SEQ ID NO: 15) D1C5 GYGSGSYWSGAFDI WGQGTMVTVSS(SEQ ID NO: 150) (SEQ ID NO: 54) D1D1 LGGESYSAD WGQGTLVTVSS(SEQ ID NO: 151) (SEQ ID NO: 13) HB8 TLGRSTVATDY WGQGTLVTVSS(SEQ ID NO: 152) (SEQ ID NO: 13) HC2 GPGAAQDY WGQGTLVTVSS(SEQ ID NO: 153) (SEQ ID NO: 13) HC4 GAVGATTAFDY WGQGTLVTVSS(SEQ ID NO: 154) (SEQ ID NO: 13) HE3 VASSSSLGMDV WGQGTTVTVSS(SEQ ID NO: 155) (SEQ ID NO: 156) HF1 SVVGATSLDY WGQGTLVTVSS(SEQ ID NO: 157) (SEQ ID NO: 13) HH3 GIVGATAFDY WGQGTLVTVSS(SEQ ID NO: 158) (SEQ ID NO: 13) E8H11 DYGYCSGGSCYSPFDY WGQGTLVTVSS(SEQ ID NO: 197) (SEQ ID NO: 13) E8H7 RLEWL-----PLAWDY WGQGTLVTVSS(SEQ ID NO: 198) (SEQ ID NO: 13) E8G12 AARIAAR---PGPLDY WGQGTLVTVSS(SEQ ID NO: 199) (SEQ ID NO: 13) E8F11 H--LSSG----SSVDY WGQGTLVTVSS(SEQ ID NO: 200) (SEQ ID NO: 13) E8C9 AGPRTTV----TTVDS WGQGTLVTVSS(SEQ ID NO: 201) (SEQ ID NO: 13) D6G9 GLKDSSG------FDY WGQGTLVTVSS(SEQ ID NO: 202) (SEQ ID NO: 13) D6D3 GIVGATA------FDY WGQGTLVTVSS(SEQ ID NO: 203) (SEQ ID NO: 13)

TABLE 2Light chain FRs and CDRs of antibodies that specifically bind CD44 cloneV_(L) FR1 V_(L) CDR1 V_(L) FR2 F2-1A6 SELTQDPAVSVALGQTVRITC QGDSLRSYYASWYQQKPGQAPLLVIY (SEQ ID NO: 16) (SEQ ID NO: 17) (SEQ ID NO: 18) F2-1H9SELTQDPAVSVALGQTVRITC QGDSLRSYYAS WYQRKPGQAPLLVIY (SEQ ID NO: 16)(SEQ ID NO: 17) (SEQ ID NO: 19) D1C5 EIVLTQSPLSLPVTPGEPASISCRSSQSLLHTNGYNYLD WYLQKPGQSPQLLIY (SEQ ID NO: 159) (SEQ ID NO: 160(SEQ ID NO: 161) D1D1 HVILTQDPAVSVALGQTVKITC QGDSLRSYYAS WYQQKPGQAPLLVLY(SEQ ID NO: 162) (SEQ ID NO: 17) (SEQ ID NO: 163) HB8SELTQDPAVSVALGQTVRITC QGDSLRSYYAS WYQQKPGQAPVLVIY (SEQ ID NO: 16)(SEQ ID NO: 17) (SEQ ID NO: 70) HC2 QSVLTQDPAVSVALGQTVTITC QGDSLRSYYASWYQQKPGQAPVLVIY (SEQ ID NO: 164) (SEQ ID NO: 17) (SEQ ID NO: 70) HC4SELTQDPAVSVALGQTVKITC QGDSLRSYYAS WYQQKPGQAPVLVIY (SEQ ID NO: 68)(SEQ ID NO: 17) (SEQ ID NO: 70) HH3 SELTQDPAVSVALGQTVRITC QGDSLRSYYASWYQQKPGQAPVLVIY (SEQ ID NO: 16) (SEQ ID NO: 17) (SEQ ID NO: 70) HF1QSVLTQDPAVSVALGQTVRITC QGDSLRDYYAS WYKQKPGQAPLLVFF (SEQ ID NO: 165)(SEQ ID NO: 166) (SEQ ID NO: 167) HE3 SELTQDPAVSVALGQTVRITC QGDSLRSYYASWYQQKPGQAPVLVIY (SEQ ID NO: 16) (SEQ ID NO: 17) (SEQ ID NO: 70) E8H11NFMLTQDPAVSVALGQTVRITC QGDSLRS---YYAS WYQQKPGQAPVLVIY (SEQ ID NO: 204)(SEQ ID NO: 17) (SEQ ID NO: 70) E8H7 S-ELTQDPAVSVALGQTVRITCQGDSLRS---YYAS WYQQKPGQAPVLVIY (SEQ ID NO: 205) (SEQ ID NO: 17)(SEQ ID NO: 70) E8G12 S-ELTQDPAVSVAVGQTVKITC QGDSLRN---YYASWYQQKPRQAPVLVIY (SEQ ID NO: 206) (SEQ ID NO: 207) (SEQ ID NO: 208) E8F11-SELTQPPSASGSPGQSVTISC TGTSSDVGGYNYVS WYQQRPGYAPKLMIY (SEQ ID NO: 209)(SEQ ID NO: 210) (SEQ ID NO: 211) E8C9 QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVH WYQQLPGTAPKLLTY (SEQ ID NO: 212) (SEQ ID NO: 213)(SEQ ID NO: 214) D6G9 NFMLTQDPAVSVALGQTVRITC QGDSLRS---YYASWYQQKPGQAPVLVIY (SEQ ID NO: 204) (SEQ ID NO: 17) (SEQ ID NO: 70) D6D3S-ELTQDPAVSVALGQTVRITC QGDSLRS---YYAS WYQQKPGQAPVLVIY (SEQ ID NO: 205)(SEQ ID NO: 17) (SEQ ID NO: 70) clone V_(L) CDR2 V_(L) FR3 F2-1A6GKNIRPS GIPDRFSGSSSGNTASLTITGAQAEDEADYYC (SEQ ID NO: 20) (SEQ ID NO: 21)F2-1H9 GKNIRPS GIPDRFSGSSSGNTASLIITGAQAEDEADYYC (SEQ ID NO: 20)(SEQ ID NO: 22) D1C5 LGSNRAS GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC(SEQ ID NO: 168) (SEQ ID NO: 169) D1D1 GENNRPSGIPDRFSGSGSGNTASLTITGAQAEDEADYYC (SEQ ID NO: 71) (SEQ ID NO: 170) HB8GKNNRPS GIPDRFSGSSSGNTASLTITGAQAEDEADYYC (SEQ ID NO: 171)(SEQ ID NO: 23) HC2 GKNNRPS GIPDRFSGSSSGNTASLTITGAQAEDEADYYC(SEQ ID NO: 171) (SEQ ID NO: 23) HC4 GKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYC (SEQ ID NO: 171) (SEQ ID NO: 23) HE3GKNNRPS GIPDRFSGSSSGNTASLTITGARAEDEADYYC (SEQ ID NO: 171)(SEQ ID NO: 172) HF1 GKSNRPS GIPNRFSGSTSGSTATLTVTGAQAEDEADYFC(SEQ ID NO: 173) (SEQ ID NO: 174) HH3 GKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYC (SEQ ID NO: 171) (SEQ ID NO: 23) E8H11GKNNRPS GIPDRFSGSSSGNTASLTITGAQAEDEADYYC (SEQ ID NO: 171)(SEQ ID NO: 23) E8H7 GKNNRPS GIPDRFSGSSSGNTASLTITGAQAEDEADYYC(SEQ ID NO: 171) (SEQ ID NO: 23) E8G12 GKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYC (SEQ ID NO: 171) (SEQ ID NO: 23) E8F11DVSNRPS GVSNRFSGSKSGNSASLDISGLQSEDEADYYC (SEQ ID NO: 215)(SEQ ID NO: 216) E8C9 GNSNRPS GVPDRFSGSKSGTSASLAITGLQAEDEADYYR(SEQ ID NO: 217) (SEQ ID NO: 218) D6C9 GKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYC (SEQ ID NO: 171) (SEQ ID NO: 23) D6D3GKNNRPS GIPDRFSGSSSGNTASLTITGAQAEDEADYYC (SEQ ID NO: 171)(SEQ ID NO: 23) clone V_(L) CDR3 V_(L) FR4 F2-1A6 NSRDSSGNHVVFGGGTKLTVLG (SEQ ID NO: 23) (SEQ ID NO: 24) F2-1H9 NSRDSSANQMFGGGTKLTVLG (SEQ ID NO: 25) (SEQ ID NO: 27) D1C5 MQGRQSPLT FGGGTKVEIKR(SEQ ID NO: 175) (SEQ ID NO: 84) D1D1 HSRDSSGKYV FGVGTKVTVLG(SEQ ID NO: 176) (SEQ ID NO: 177) HB8 NSRDSSGNPHVV FGGGTKLTVLG(SEQ ID NO: 178) (SEQ ID NO: 27) HC2 NSRDSSGNHVV FGGGTKVTVLG(SEQ ID NO: 26) (SEQ ID NO: 80) HC4 NSRDSSGNHLV FGGGTKLTVLG(SEQ ID NO: 179) (SEQ ID NO: 27) HE3 TSRDSSGKQLV FGGGTKLTVLG(SEQ ID NO: 180) (SEQ ID NO: 27) HF1 SSRDSSGRLIL FGGGTKLTVLG(SEQ ID NO: 181) (SEQ ID NO: 27) HH3 NSRDSSGNSVV FGGGTKVTVLG(SEQ ID NO: 182) (SEQ ID NO: 80) E8H11 HSRDSSGN-YL FGGGTKLTVLG(SEQ ID NO: 219) (SEQ ID NO: 27) E8H7 NSRDSSGNHKV FGGGTKLTVLG(SEQ ID NO: 220) (SEQ ID NO: 27) E8G12 NSRDRSNNHLL FGGGTKLTVLG(SEQ ID NO: 221) (SEQ ID NO: 27) E8F11 AAWDDSLREFL FGTGTKVTVLG(SEQ ID NO: 222) (SEQ ID NO: 223) E8C9 SAWDSSLFNWV FGGGTKLTVLG(SEQ ID NO: 224) (SEQ ID NO: 27) D6G9 NSRDSSAKRVV FGGGTKVTVLG(SEQ ID NO: 225) (SEQ ID NO: 80) D6D3 NSRDSSGNSVV FGGGTKVTVLG(SEQ ID NO: 182) (SEQ ID NO: 80)

Anti-EphA2

Antibodies of the present disclosure include those that specificallybind EphA2. Anti-EphA2 antibodies encompass those that competitivelybind to an epitope (e.g. on the extracellular domain) of EphA2 with 2D6,D2-1A7, D2-1A9, D2-1B1, A3H9, A3G3, A3D10, A3D1, A3C8, 1A3, 1A5, 1A8,1A12, 1B2, 1C2, 1C7, 1D8, or 15H11. The epitope bound by anti-EphA2antibodies reside in a contiguous amino acid sequence of EphA2 fromresidue position 25 to 534, as set forth below.

(SEQ ID NO: 26) QGKEVVLLDFAAAGGELGWLTHPYGKGWDLMQNIMNDMPIYMYSVCNVMSGDQDNWLRTNWVYRGEAERIFIELKFTVRDCNSFPGGASSCKETFNLYYAESDLDYGTNFQKRLFTKIDTIAPDEITVSSDFEARHVKLNVEERSVGPLTRKGFYLAFQDIGACVALLSVRVYYKKCPELLQGLAHFPETIAGSDAPSLATVAGTCVDHAVVPPGGEEPRMHCAVDGEWLVPIGQCLCQAGYEKVEDACQACSPGFFKFEASESPCLECPEHTLPSPEGATSCECEEGFFRAPQDPASMPCTRPPSAPHYLTAVGMGAKVELRWTPPQDSGGREDIVYSVTCEQCWPESGECGPCEASVRYSEPPHGLTRTSVTVSDLEPHMNYTFTVEARNGVSGLVTSRSFRTASVSINQTEPPKVRLEGRSTTSLSVSWSIPPPQQSRVWKYEVTYRKKGDSNSYNVRRTEGFSVTLDDLAPDTTYLVQVQALTQEGQGAGSKVHEFQTLSPEGSGN

The residue position numbers of EphA2 are determined based on thesequence set forth in GenBank Accession No. NP_(—)004422.2 or UniProtAccession No. P29317.

Anti-EphA2 antibodies also encompass those that competitively bind tothe ligand binding site of EphA2. For example, antibodies 2D6, D2-1A7,and D2-1A9 compete with Ephrin A1, the natural ligand of EphA2, forbinding to EphA2. Thus, the subject antibodies can also compete withEphrin A1 for binding to an epitope of EphA2.

Antigens that comprise epitopes that are similar to the epitopes ofEphA2 can also be binding targets of subject antibodies. When bound toan antigen on a cell surface (e.g. EphA2), certain of the subjectantibodies will be internalized by the cell.

Epitopes for which anti-EphA2 antibodies have affinity are cell-surfaceexposed and solvent-accessible on many cancer cells, particularly on theplasma membrane of cells. The epitopes can be accessible to the subjectantibodies when the cells are live. The cancer cells can include thosederived from tissue of epithelial in origin. For example, the epitopescan be found on cancer cells derived from breast cancers, skin cancers(e.g. melanoma), lung cancers, prostate cancers, colon cancers, and/orovarian cancers. Other cancers cells for which anti-EphA2 antibodieshave affinity may be liver cancer, esophageal squamous cell carcinoma,epidermoid cancer, pancreatic cancer, glioblastomas, neuroblastomas,and/or other neural cancers, for example.

Additional examples of subject antibodies encompass those that have thesame binding specificities and contain at least two CDRs that eachindependently shares at least about 80%, at least about 87%, at leastabout 93%, at least about 94%, or up to 100% amino acid sequenceidentity with the amino acid sequence of a V_(H) CDR of antibodies shownin FIGS. 8, 46, and 47, and in tables below (e.g. V_(H) CDR1 of 2D6; orV_(H) of CDR1 of A3H9, A3G3, A3D10, A3D1, A3C8, 1A3, 1A5, 1A8, 1A12,1B2, 1C2, 1C7, 1D8, or 15E11). The subject antibody can also include allthree CDRs from any V_(H) of any antibody shown in FIGS. 8, 46, and 47,such that each V_(H) CDR in the subject antibody is selected from asingle antibody shown in FIG. 8, 46, or 47 or tables below (e.g., Table3) and each V_(H) CDR independently shares at least about 80%, at leastabout 87%, at least about 93%, at least about 94%, or up to 100% aminoacid sequence identity with the amino acid sequence of the correspondingV_(H) CDR of an antibody shown in FIGS. 8, 46, and 47 or in tablesbelow. For example, the heavy chain of a subject antibody can contain atleast two V_(H) CDRs or all three V_(H) CDRs of 2D6. Alternatively, theheavy chain can contain at least two V_(H) CDRs or all three V_(H) CDRsof D2-1A7. Other examples include a heavy chain that contains at leasttwo V_(H) CDRs or all three V_(H) CDRs of D2-1A9 and a heavy chain thatcontains at least two V_(H) CDRs or all three V_(H) CDRs of D2-1B1. Asfurther examples, the heavy chain of a subject antibody can contain atleast two V_(H) CDRs or all three V_(H) CDRs of A3H9, A3G3, A3D10, A3D1,A3C8, 1A3, 1A5, 1A8, 1A12, 1B2, 1C2, 1C7, 1D8, or 15H11.

Similarly for the light chain, a subject antibody can contain at leasttwo CDRs that are each independently at least about 80%, at least about87%, at least about 93%, at least about 94%, or up to 100% amino acidsequence identity with the amino acid sequence of a V_(L) CDR of anantibody shown in FIG. 8, 46, or 47 or tables below (e.g. V_(L) CDR1 of2D6; or V_(L) of CDR1 of A3H9, A3G3, A3D10, A3D1, A3C8, 1A3, 1A5, 1A8,1A12, 1B2, 1C2, 1C7, 1D8, or 15E11). The subject antibody can also oralternatively include all three V_(L) CDRs from any of antibodies shownin FIGS. 8, 46, and 47 or tables below (e.g., Table 4) and each V_(L)CDR independently shares at least about 80%, at least about 87%, atleast about 93%, at least about 94%, or up to 100% amino acid sequenceidentity with the amino acid sequence of a V_(L) CDR of an antibodyshown in FIG. 8, 46, or 47 or tables below. For example, the light chainof a subject antibody can contain two V_(L) CDRs or all three V_(L) CDRsof 2D6. Alternatively, the light chain can contain two V_(L) CDRs or allthree V_(L) CDRs of D2-1A7. Other examples include a light chain thatcontains two V_(L) CDRs or all three V_(L) CDRs of D2-1A9 and a lightchain that contains two V_(L) CDRs or all three V_(L) CDRs of D2-1B1. Asfurther examples, the light chain of a subject antibody can contain twoV_(L) CDRs or all three V_(L) CDRs of A3H9, A3G3, A3D10, A3D1, A3C8,1A3, 1A5, 1A8, 1A12, 1B2, 1C2, 1C7, 1D8, or 15H11.

Optionally, antibodies can contain the exactly the same (i.e. 100%identity), similar, or different framework sequences (FR) in any ofcorresponding framework sequences in the heavy or light chain providedin FIGS. 8, 46, and 47 or tables below (e.g., Tables 3 and 4). Where theframework sequences are similar, the framework may be at least about85%, at least about 86%, at least about 90%, at least about 93%, atleast about 96%, at least about 98%, or up to 100% identity to acorresponding framework sequence in any of antibodies shown in FIGS. 8,46, and 47 or tables below.

An antibody of the present disclosure may therefore contain afull-length V_(H) and/or full length V_(L) sequence that is at least 80%identity, at least 85%, at least 90%, at least 95%, up to 100% aminoacid sequence identity to a full-length V_(H) or V_(L) sequence shown inFIG. 8, 46, or 47 or tables below. For example, a subject antibody cancontain the full length V_(H) and/or full length V_(L) of 2D6.Alternatively, the subject antibody can contain the full length V_(H)and/or full length V_(L) of D2-1A7. Other examples include an antibodycontaining the full length V_(H) and/or full length V_(L) of D2-1A9 andan antibody containing the full length V_(H) and/or full length V_(L) ofD2-1B1. Further examples include an antibody containing the full lengthV_(H) and/or full length V_(L) of A3H9, A3G3, A3D10, A3D1, A3C8, 1A3,1A5, 1A8, 1A12, 1B2, 1C2, 1C7, 1D8, or 15H11. Examples of antibodies ofthe present disclosure that bind to EphA2 are listed in tables below.

TABLE 3Heavy chain FRs and CDRs of antibodies that specifically bind EphA2clone V_(H) FR1 V_(H) CDR1 V_(H) FR2 2D6 QVQLQESGGGLVQPGGSLRLSCAASGFTFSSYAMS WVRQAPGKGLDWVS (SEQ ID NO: 5) (SEQ ID NO: 32) (SEQ ID NO: 27)D2-1A7 QVQLQQSGGGVVQPGRSLRLSCAASGFTFS SYAMH WVRQAPGKGLEWVA(SEQ ID NO: 28) (SEQ ID NO: 29) (SEQ ID NO: 7) D2-1A9QVQLVESGGGLIQPGGSLKLSCAASGFTVS NSYMS WVRQAPGKGLEWVA (SEQ ID NO: 30)(SEQ ID NO: 31) (SEQ ID NO: 7) D2-1B1 QVQLQESGGGVVQPGRSLRLSCAASGFTFSSYGMH WVRQAPGKGLEWVA (SEQ ID NO: 32) (SEQ ID NO: 33) (SEQ ID NO: 7) A3H9QVLLVESGGGVVQAGASLRVSCAASGFSLT SYGMH WVRQAPGKGLEWVA (SEQ ID NO: 126)(SEQ ID NO: 41) (SEQ ID NO: 7) A3G3 QVQLVESGGGVVQPGRSLRLSCAASGFTFS SYAMGWVRQAPGKGLEWVA (SEQ ID NO: 131) (SEQ ID NO: 189) (SEQ ID NO: 7) A3D10LVQLVQSGGGLVKPGGSLRLSCAASGFTFS TYSMN WVRQAPGKGLEWVS (SEQ ID NO: 226)(SEQ ID NO: 227) (SEQ ID NO: 133) A3D1 QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYGMH WVRQAPGKGLEWVA (SEQ ID NO: 185) (SEQ ID NO: 41) (SEQ ID NO: 7)A3C8 QLQLVESGGGLVQPGGSLRLSCAASGFTFS SYAMN WVRQAPGKGLEWVS(SEQ ID NO: 228) (SEQ ID NO: 229) (SEQ ID NO: 133) 1A3QVQLQQSGPGLVKPSQTLSLTCAISGDSVS SNSAA WNWIRQSPSRGLEWLG (SEQ ID NO: 230)(SEQ ID NO: 231) (SEQ ID NO: 232) 1A5 QVQLQESGGGVVQPGGSLRLSCAASGFTFSNYAMH WVRQAPGKGLEYVS (SEQ ID NO: 233) (SEQ ID NO: 234) (SEQ ID NO: 184)1A8 QVQLQQSGGGLVQPGGSLRLSCSASGFTFS NYAIH WVRQAPGKGLEYVS (SEQ ID NO: 235)(SEQ ID NO: 236) (SEQ ID NO: 184) 1A12 QVQLQESGGGLVQPGGFLRLSCAASGFTFSSYGMH WVRQTPGKGLEWVS (SEQ ID NO: 237) (SEQ ID NO: 41) (SEQ ID NO: 238)1B2 QVQLVESGGGVVQPGRSLRLSCAASGFTFS SYGMH WVRQAPGKGLEWVA (SEQ ID NO: 131)(SEQ ID NO: 41) (SEQ ID NO: 7) 1C2 QVQLQESGGGLVKPGGSLRLSCAGSGFIFN TYSMNWVRQSPGKGLEWVS (SEQ ID NO: 239) (SEQ ID NO: 227) (SEQ ID NO: 240) 1C7QVQLVESGGGVVQPGRSLRLSCAASGFTFS SYGMH WVRQAPGKGLEWVS (SEQ ID NO: 131)(SEQ ID NO: 41) (SEQ ID NO: 133) 1D8 QVQLVESGGGLIQPGGSLRLSCAASGFTVSSNYMS WVRQAPGKGLEWVS (SEQ ID NO: 241) (SEQ ID NO: 242) (SEQ ID NO: 133)clone V_(H) CDR2 V_(H) FR3 2D6 IIYNGDNTYYADSVKGRFTISRDNSKNSLYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 34) (SEQ ID NO: 35) D2-1A7VISYDGSNKYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 36)(SEQ ID NO: 37) D2-1A9 VIYSAGNTYYADSVKG RFTISRDTSNNTVHLQMNSLRPEDTAVYYCAR(SEQ ID NO: 38) (SEQ ID NO: 39) D2-1B1 VISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK (SEQ ID NO: 45) (SEQ ID NO: 40) A3H9FISSDGSDKYYVDSVKG RFTISRDTSKNMMYLQMNSLTTEDTAVYYCAK (SEQ ID NO: 243)(SEQ ID NO: 244) A3G3 VIYRDGHGYYADSVKG RFTVSRDSSENTVYLQMNSLRAEDTAIYYCAS(SEQ ID NO: 245) (SEQ ID NO: 246) A3D10 SISSSSSYIYYADSVKGRFTISRDNANNSLYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 247) (SEQ ID NO: 248) A3D1VIWYDGSNKYYADSVKG RFTISRDNSRNTLYLEMNSLRAEDTAVYYCVK (SEQ ID NO: 249)(SEQ ID NO: 250) A3C8 AISGSGGNTYYADSVKG RFTISRDNSNNALYLQMNSLRVEDTAVYYCAR(SEQ ID NO: 251) (SEQ ID NO: 252) 1A3 RTYYRSKWYNDYAVSVKSRITINPDTSKNLFSLQLNSVTPEDTALYYCAR (SEQ ID NO: 253) (SEQ ID NO: 254) 1A5SISSNGGGTYYADSVKG RFTISRDDAKNTLYLQLNSLRDEDTAVYYCAK (SEQ ID NO: 255)(SEQ ID NO: 256) 1A8 AINSNGGSTYYADSVKG RSIISRDNSMNTVYLQMSSLRAEDTAVYYCVK(SEQ ID NO: 257) (SEQ ID NO: 258) 1A12 YISSSSSYTNYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 259) (SEQ ID NO: 46) 1B2VISYDGSNKYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 45)(SEQ ID NO: 46) 1C2 STGGSGKNTFYADSVRG RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR(SEQ ID NO: 260) (SEQ ID NO: 261) 1C7 YISSSGSYTNYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCAK (SEQ ID NO: 262) (SEQ ID NO: 263) 1D8VIYSGGSTYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 264)(SEQ ID NO: 46) clone V_(H) CDR3 V_(H) FR4 2D6 WSGTSYDY WGQGTLVTVSS(SEQ ID NO: 41) (SEQ ID NO: 13) D2-1A7 ASVGATGPFDI WGQGTMVTVSS(SEQ ID NO: 42) (SEQ ID NO: 43) D2-1A9 EGSFGYDY RGQGTLVTVSS(SEQ ID NO: 44) (SEQ ID NO: 45) D2-1B1 VIAGGAYYGSADY WGQGTLVTVSS(SEQ ID NO: 46) (SEQ ID NO: 13) A3H9 DWGSNWYLFDY WGQGTLVTVSS(SEQ ID NO: 265) (SEQ ID NO: 13) A3G3 HDYAGNPAGSASGY WGQGTLVTVSS(SEQ ID NO: 266) (SEQ ID NO: 13) A3D10 GNTVAQRLDVFDY WGQGTLVTVSS(SEQ ID NO: 267) (SEQ ID NO: 13) A3D1 DRQPDGRWPFDL WGQGTLVTVSS(SEQ ID NO: 268) (SEQ ID NO: 13) A3C8 DASYYADDY WGQGTLVTVSS(SEQ ID NO: 269) (SEQ ID NO: 13) 1A3 EEDYSGFQH WGQGTLVTVSS(SEQ ID NO: 270) (SEQ ID NO: 13) 1A5 DYFGSIDY WGQGTLVTVSS(SEQ ID NO: 271) (SEQ ID NO: 13) 1A8 EENGSGFDS WGQGTLVTVSS(SEQ ID NO: 272) (SEQ ID NO: 13) 1A12 DNWYFDL WGRGTLVTVSS(SEQ ID NO: 273) (SEQ ID NO: 274) 1B2 ALYYDEALDY WGQGTLVTVSS(SEQ ID NO: 275) (SEQ ID NO: 13) 1C2 EDSSGSFDY WGQGTLVTVSS(SEQ ID NO: 276) (SEQ ID NO: 13) 1C7 VRGWDGDYLDY WGQGTLVTVSS(SEQ ID NO: 277) (SEQ ID NO: 13) 1D8 GGFSGYDYFDY WGQGTLVTVSS(SEQ ID NO: 278) (SEQ ID NO: 13)

TABLE 4Light chain FRs and CDRs of antibodies that specifically bind EphA2clone V_(L) FR1 V_(L) CDR1 V_(L) FR2 2D6 SELTQDPAVSVALGQTVRITCQGDSLRSYYAS WYQQKPGQAPLLVIY (SEQ ID NO: 16) (SEQ ID NO: 17)(SEQ ID NO: 18) D2-1A7 SELTQDPAVSVALGQTVSITC QGDSLRSYYAS WYQQKPGQAPLLVIY(SEQ ID NO: 47) (SEQ ID NO: 17) (SEQ ID NO: 18) D2-1A9DIVMTQSPGTLSLSPGERATLSC RASQSVSSSFLG WYQQKPGQAPRLLIY (SEQ ID NO: 48)(SEQ ID NO: 49) (SEQ ID NO: 50) D2-1B1 SELTQDPAVSVALGQTVKITC QGDSLRTYYASWYQQKPGQAPVLVIY (SEQ ID NO: 51) (SEQ ID NO: 52) (SEQ ID NO: 53) A3H9SVLTQPPSASETPGQRVTISC SGSSSNIGANTVH WYQQFPGTAPKLLIY (SEQ ID NO: 296)(SEQ ID NO: 297) (SEQ ID NO: 298) A3G3 SALTQPASVSGSPGQSVTISCTGTSSDVGGYDYVS WYQQHPGKAPKLVMY (SEQ ID NO: 299) (SEQ ID NO: 300)(SEQ ID NO: 301) A3D10 SVLTQPPSVSGAPGQRVTISC TGGSSNVGAGFDVHWYQQLPGTAPKLLIY (SEQ ID NO: 302) (SEQ ID NO: 303) (SEQ ID NO: 304) A3D1SALTQPASVSGSPGQSVTISC TGANSDLGGYNYVS WYQHHPAKAPKLIIY (SEQ ID NO: 305)(SEQ ID NO: 306) (SEQ ID NO: 307) A3C8 SELTQDPAVSVALGQTVRITC QGDSLKSYYASWYQQKPGQAPVLVIY (SEQ ID NO: 16) (SEQ ID NO: 308) (SEQ ID NO: 70) 1A3QSALTQPASVSGSPGQSITISC TGTSSDVGGYNYVS WYQQHPGKAPKLMIY (SEQ ID NO: 309)(SEQ ID NO: 210) (SEQ ID NO: 211) 1A5 PELTQDPAVSVALGQTVTITC QGDSLRSYYASWYQQKPGQAPLLVIY (SEQ ID NO: 212) (SEQ ID NO: 17) (SEQ ID NO: 18) 1A8EIVLTQSPSSVSASVGDRVTITC RASQDISKWLA WYQQRPGKVPRLLIY (SEQ ID NO: 213)(SEQ ID NO: 214) (SEQ ID NO: 215) 1A12 DVVMTQSPSTLSASVGDRVSITCRASESISRWLA WYQQKPGKAPKALIY (SEQ ID NO: 216) (SEQ ID NO: 217)(SEQ ID NO: 218) 1B2 EIVLTQSPSSLSASVGDRVTITC RASQDMSRWLA WYQQKPGKAPKLLIH(SEQ ID NO: 219) (SEQ ID NO: 220) (SEQ ID NO: 221) 1C2DIQMTQSPSTLSASIGDRVTITC RASEGIYHWLA WYQQKPGKAPKLLIY (SEQ ID NO: 222)(SEQ ID NO: 223) (SEQ ID NO: 224) 1C7 DIQMTQSPSLLSASVGDRVTITCRASQGINNYLA WYQQKPGKAPKLLIY (SEQ ID NO: 225) (SEQ ID NO: 226)(SEQ ID NO: 224) 1D8 QSVLTQPPSVSGAPGQRVTISC TGSSSNIGAGYDVHWYQQLPGTAPKLLIY (SEQ ID NO: 212) (SEQ ID NO: 213) (SEQ ID NO: 304) cloneV_(L) CDR2 V_(L) FR3 2D6 GENNRPS GIPDRFSGSSSGNTASLTITGAQAEDEADYYC(SEQ ID NO: 54) (SEQ ID NO: 23) D2-1A7 GENNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYC (SEQ ID NO: 71) (SEQ ID NO: 23) D2-1A9GASSRAT GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC (SEQ ID NO: 55) (SEQ ID NO: 56)D2-1B1 GENSRPS GIPDRFSGSSSGNTASLTITGAQAEDEADYYC (SEQ ID NO: 57)(SEQ ID NO: 23) A3H9 SYSQRPS GVPDRFSDSKSGTSASLAISGLQSEDEADYYC(SEQ ID NO: 404) (SEQ ID NO: 405) A3G3 SHNQRSSGVPDRFSGSKSGNSASLDISGLQSEDEADYYR (SEQ ID NO: 406) (SEQ ID NO: 407) A3D10GDKNRPS GVPDRFSGSRSGTSASLAITGLQAEDEADYYC (SEQ ID NO: 408)(SEQ ID NO: 409) A3D1 EVNNRPS GVSHRFSGSKSANTASLTISGLQAEDEADYYC(SEQ ID NO: 410) (SEQ ID NO: 411) A3C8 GKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYC (SEQ ID NO: 171) (SEQ ID NO: 23) 1A3EGSKRPS GVPERFSGSNSGNTATLTIGRVEAGDEADYYC (SEQ ID NO: 412)(SEQ ID NO: 413) 1A5 GKNIRPS GIPDRFSGSSSGNSASLTITGAQAEDEADYYC(SEQ ID NO: 22) (SEQ ID NO: 414) 1A8 SASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFASYFC (SEQ ID NO: 415) (SEQ ID NO: 416) 1A12KASSLES GVPSRFSGSGSATEFTLTINSLQPDDFATYYC (SEQ ID NO: 417)(SEQ ID NO: 418) 1B2 SASTLQS GVPSRFSGSGSGTEFTLTISSLQPEDFATYYC(SEQ ID NO: 419) (SEQ ID NO: 420) 1C2 KASSLASGAPSRFSGSGSGTDFTLTISSLQPDDFATYYC (SEQ ID NO: 421) (SEQ ID NO: 422) 1C7AASTLQS GVPSRFSGSGSGTEFTLTISGLQPEDFATYYC (SEQ ID NO: 423)(SEQ ID NO: 424) 1D8 VNSNRPS GVPDRFSGSKSGTSASLAITGLQAEDEADYYC(SEQ ID NO: 425) (SEQ ID NO: 426) clone V_(L) CDR3 V_(L) FR4 2D6HSRDSSGTHLRV FGGGTKVTVLG (SEQ ID NO: 58) (SEQ ID NO: 59) D2-1A7NSRDSSGTHLTV FGGGTKLTVLG (SEQ ID NO: 60) (SEQ ID NO: 27) D2-1A9QQYGISPLT FGGGTKVEIKR (SEQ ID NO: 61) (SEQ ID NO: 62) D2-1B1HSRDSSGTHLRV FGGGTKLTVLG (SEQ ID NO: 79) (SEQ ID NO: 27) A3H9AAWDDILNGWV FGGGTKVTVLG (SEQ ID NO: 279) (SEQ ID NO: 80) A3G3AAWDDSLSEFL FGTGTKLTVLG (SEQ ID NO: 280) (SEQ ID NO: 281) A3D10QAYDSSLRGSV FGGGTKLTVLG (SEQ ID NO: 282) (SEQ ID NO: 27) A3D1 SSYRSGGTYVFGTGTKLTVLG (SEQ ID NO: 283) (SEQ ID NO: 281) A3C8 HSRDSSGNHPVVFGGGTKVTVLG (SEQ ID NO: 284) (SEQ ID NO: 80) 1A3 QAWDSTSDHVV FGGGTKVTVLG(SEQ ID NO: 285) (SEQ ID NO: 80) 1A5 HSRDSSGTHLRV FGGGTKLTVLG(SEQ ID NO: 79) (SEQ ID NO: 27) 1A8 QQASVFPVT FGGGTKLEIKR(SEQ ID NO: 286) (SEQ ID NO: 287) 1A12 QQYSSYPLT FGQGTKVDIKR(SEQ ID NO: 288) (SEQ ID NO: 289) 1B2 QQLGVYPLT IGGGTKVEIKR(SEQ ID NO: 290) (SEQ ID NO: 291) 1C2 QQYHTISRT FGPXTKLEIKR(SEQ ID NO: 292) (SEQ ID NO: 293) 1C7 QNLNSYPLT FGGGTKVEIKR(SEQ ID NO: 294) (SEQ ID NO: 84) 1D8 QSYDSSLSGWV FGGGTKLTVLG(SEQ ID NO: 295) (SEQ ID NO: 27)

It will be appreciated that the amino acid sequence of a CDR can also bedefined using alternative systems, which will be readily apparent to andapplied by the ordinarily skilled artisan (see, “Sequences of Proteinsof Immunological Interest,” E. Kabat et al., U.S. Department of Healthand Human Services, (1991 and Lefranc et al. IMGT, the internationalImMunoGeneTics information system. Nucl. Acids Res., 2005, 33,D593-D597)). A detailed discussion of the IMGTS system, including howthe IMGTS system was formulated and how it compares to other systems, isprovided on the World Wide Web atwww(dot)imgt.cines.fr/textes/IMGTScientificChart/Numbering/IMGTnumberingsTable.html.All amino acid sequences of CDR in the present disclosure are definedaccording to Kabat et al., supra, unless otherwise indicated.

The variable chains disclosed herein can be joined directly or through alinker (e.g., (Gly₄Ser)₃, SEQ ID NO: 63) to form a single-chainantibody. Details on linkers are discussed later below.

Method of Antibody Production

Using the information provided herein, the anti-CD44 and anti-EphA2antibodies of the present disclosure are prepared using standardtechniques well known to those of skill in the art.

For example, the nucleic acid sequences provided in FIG. 9 can be usedto express the subject antibodies. Alternatively, the polypeptidesequences provided herein (see, e.g., tables above and/or FIGS. 7, 8, 9,and 44-47) can be used to determine appropriate nucleic acid sequencesencoding the antibodies and the nucleic acids sequences then used toexpress one or more antibodies specific for EphA2 or CD44. The nucleicacid sequence(s) can be optimized to reflect particular codon“preferences” for various expression systems according to standardmethods well known to those of skill in the art.

Using the sequence information provided, the nucleic acids may besynthesized according to a number of standard methods known to those ofskill in the art. Oligonucleotide synthesis, is preferably carried outon commercially available solid phase oligonucleotide synthesis machines(Needham-VanDevanter et al. (1984) Nucleic Acids Res. 12:6159-6168) ormanually synthesized using, for example, the solid phase phosphoramiditetriester method described by Beaucage et. al. (1981) Tetrahedron Letts.22(20): 1859-1862.

Once a nucleic acid encoding a subject antibody is synthesized it can beamplified and/or cloned according to standard methods. Molecular cloningtechniques to achieve these ends are known in the art. A wide variety ofcloning and in vitro amplification methods suitable for the constructionof recombinant nucleic acids are known to persons of skill in the artand are the subjects of numerous textbooks and laboratory manuals.

Expression of natural or synthetic nucleic acids encoding the antibodiesof the present disclosure can be achieved by operably linking a nucleicacid encoding the antibody to a promoter (which is either constitutiveor inducible), and incorporating the construct into an expression vectorto generate a recombinant expression vector. The vectors can be suitablefor replication and integration in prokaryotes, eukaryotes, or both.Typical cloning vectors contain functionally appropriately orientedtranscription and translation terminators, initiation sequences, andpromoters useful for regulation of the expression of the nucleic acidencoding the antibody. The vectors optionally contain generic expressioncassettes containing at least one independent terminator sequence,sequences permitting replication of the cassette in both eukaryotes andprokaryotes, e.g., as found in shuttle vectors, and selection markersfor both prokaryotic and eukaryotic systems.

To obtain high levels of expression of a cloned nucleic acid it iscommon to construct expression plasmids which typically contain a strongpromoter to direct transcription, a ribosome binding site fortranslational initiation, and a transcription/translation terminator,each in functional orientation to each other and to the protein-encodingsequence. Examples of regulatory regions suitable for this purpose in E.coli are the promoter and operator region of the E. coli tryptophanbiosynthetic pathway, the leftward promoter of phage lambda (P_(L)), andthe L-arabinose (araBAD) operon. The inclusion of selection markers inDNA vectors transformed in E. coli is also useful. Examples of suchmarkers include genes specifying resistance to ampicillin, tetracycline,or chloramphenicol. Expression systems for expressing antibodies areavailable using, for example, E. coli, Bacillus sp. and Salmonella. E.coli systems may also be used.

The antibody gene(s) may also be subcloned into an expression vectorthat allows for the addition of a tag (e.g., FLAG, hexahistidine, andthe like) at the C-terminal end or the N-terminal end of the antibody(e.g. scFv) to facilitate purification. Methods of transfecting andexpressing genes in mammalian cells are known in the art. Transducingcells with nucleic acids can involve, for example, incubating lipidicmicroparticles containing nucleic acids with cells or incubating viralvectors containing nucleic acids with cells within the host range of thevector. The culture of cells used in the present disclosure, includingcell lines and cultured cells from tissue (e.g., tumor) or blood samplesis well known in the art.

Once the nucleic acid encoding a subject antibody is isolated andcloned, one can express the nucleic acid in a variety of recombinantlyengineered cells known to those of skill in the art. Examples of suchcells include bacteria, yeast, filamentous fungi, insect (e.g. thoseemploying baculoviral vectors), and mammalian cells.

Isolation and purification of a subject antibody can be accomplishedaccording to methods known in the art. For example, a protein can beisolated from a lysate of cells genetically modified to express theprotein constitutively and/or upon induction, or from a syntheticreaction mixture, by immunoaffinity purification (or precipitation usingProtein L or A), washing to remove non-specifically bound material, andeluting the specifically bound antibody. The isolated antibody can befurther purified by dialysis and other methods normally employed inprotein purification methods. In one embodiment, the antibody may beisolated using metal chelate chromatography methods. Antibodies of thepresent disclosure may contain modifications to facilitate isolation, asdiscussed above.

The subject antibodies may be prepared in substantially pure or isolatedform (e.g., free from other polypeptides). The protein can present in acomposition that is enriched for the polypeptide relative to othercomponents that may be present (e.g., other polypeptides or other hostcell components). Purified antibodies may be provided such that theantibody is present in a composition that is substantially free of otherexpressed proteins, e.g., less than 90%, usually less than 60% and moreusually less than 50% of the composition is made up of other expressedproteins.

The antibodies produced by prokaryotic cells may require exposure tochaotropic agents for proper folding. During purification from E. coli,for example, the expressed protein can be optionally denatured and thenrenatured. This can be accomplished, e.g., by solubilizing thebacterially produced antibodies in a chaotropic agent such as guanidineHCl. The antibody is then renatured, either by slow dialysis or by gelfiltration. Alternatively, nucleic acid encoding the antibodies may beoperably linked to a secretion signal sequence such as pelB so that theantibodies are secreted into the periplasm in correctly-folded form.

The present disclosure also provides cells that produce subjectantibodies, where suitable cells include eukaryotic cells, e.g.,mammalian cells. The cells can be a hybrid cell or “hybridoma” that iscapable of reproducing antibodies in vitro (e.g. monoclonal antibodies,such as IgG). For example, the present disclosure provides a recombinanthost cell (also referred to herein as a “genetically modified hostcell”) that is genetically modified with one or more nucleic acidscomprising nucleotide sequence encoding a subject antibody.

Techniques for creating recombinant DNA versions of the antigen-bindingregions of antibody molecules which bypass the generation of hybridomasare also contemplated herein. DNA is cloned into a bacterial (e.g.,bacteriophage), yeast (e.g. Saccharomyces or Pichia) insect or mammalianexpression system, for example. One example of a suitable technique usesa bacteriophage lambda vector system having a leader sequence thatcauses the expressed antibody (e.g. Fab or scFv) to migrate to theperiplasmic space (between the bacterial cell membrane and the cellwall) or to be secreted. One can rapidly generate a great numbers offunctional fragments (e.g. scFv) for those which bind the tumorassociated antigen.

Modification

The present disclosure encompasses antibodies and nucleic acids that aremodified to provide a desired feature, e.g., to facilitate delivery to aspecific type of tissue and/or cells in a subject, to increase serumhalf-life, to supplement anti-cancer activity, etc. The antibodies ofthe present disclosure can be provided with or without modification, andinclude human antibodies humanized antibodies and chimeric antibodies.One way to modify a subject antibody is to conjugate (e.g. link) one ormore additional elements at the N- and/or C-terminus or to any aminoacid (including an internal amino acid) of the antibody, where suchadditional elements include, e.g., another protein and/or a drug orcarrier molecule.

A subject antibody modified with one or more additional elements retainsthe desired binding specificity, while exploiting properties of the oneor more additional elements to impart an additional desiredcharacteristic. For example, a subject antibody can be conjugated to asecond molecule that aids in solubility, storage or other handlingproperties, cell permeability, half-life, reduction in immunogenicity,controls release and/or distribution such as by targeting a particularcell (e.g., neurons, leucocytes, tumor cells, etc.) or cellular location(e.g., lysosome, endosome, mitochondria etc.), tissue or other bodilylocation (e.g., blood, neural tissue, particular organs etc.). Otherexamples include the conjugation of a dye, fluorophore or otherdetectable labels or reporter molecules for assays, tracking, imaging,and the like. More specifically, a subject antibody can be conjugated toa second molecule such as a peptide, polypeptide, dye, fluorophore,nucleic acid, carbohydrate, anti-cancer agent, lipid, radionuclide, andthe like (e.g., at either the reducing or non-reducing end), such as theattachment of a lipid moiety, including N-fatty acyl groups such asN-lauroyl, N-oleoyl, fatty amines such as dodecyl amine, oleoyl amine,and the like.

For example, for antibodies that can be internalized into cells, theantibody or nucleic acids of the present disclosure may be furthermodified to increase or decrease the efficiency of delivery into cells.Gene delivery methods are also contemplated herein to deliver nucleicacids that direct expression of proteins (e.g., a subject antibody orother protein) in cells. The efficiency of cellular uptake (e.g.endocytosis) of antibodies can be increased or decreased by linking topeptides or proteins. For example, a given antibody can be linked to aligand for a target receptor or large molecule that is more easilyengulfed by endocytotic mechanisms, such as another antibody, where suchan antibody is an “antibody conjugate.” The conjugate payload (e.g., aligand) can also be released by acid hydrolysis or enzymatic activitywhen the endocytotic vesicle fuses with lysosomes. To modify cellularuptake, the conjugate can include a ligand that retains the antibody onthe surface of a cell, which can be useful as a control for (e.g., todecrease) cellular uptake, or in some instances decrease uptake in onecell type while increasing it in others.

Where the antibody is linked to another antibody, the antibody may bebispecific. As noted above, bispecific antibodies refer to antibodiesthat are specific for two different epitopes that may or may not be ofthe same antigen.

Other features of a conjugated antibody may include one where theconjugate reduces toxicity relative to unconjugated antibody. Anotherfeature is that the conjugate may target a type of cell or organ (e.g.cancerous cell or cancerous tissue) more efficiently than anunconjugated antibody.

Additional examples include an antibody conjugated with one or moremolecules that complement, potentiate, enhance or can otherwise operatesynergistically in connection with the antibody. The antibody can haveattached an anti-cancer drug, e.g., for delivery to a site of a cancerto further facilitate cell killing or clearance, e.g., ananti-proliferation moiety (e.g., VEGF antagonist, e.g., an anti-VEGFantibody), a toxin (e.g., ricin, Pseudomonas exotoxin A, and the like),a radionuclide (e.g. ⁹⁰Y, ¹³¹I, ¹⁷⁷L, ¹⁰B for boron neutron capture, andthe like), an anti-cancer agent (see Table 5, below), and/or anoligonucleotide (e.g. siRNA).

For example, an antibody may be formulated in a lipidic nanoparticle(e.g., a liposome) by covalent or non-covalent modifications. Theantibody may be attached to the surface of a lipidic nanoparticledirectly via an Fc region, for example. The antibody may also becovalently attached to a terminus of a polymer grafted at or insertedinto the surface of a lipidic nanoparticle via a linker. Such conjugatedlipidic nanoparticles may be referred to herein as “immunoliposomes”.The subject antibodies in an immunoliposome can act as a targetingmoiety enabling the immunoliposomes to specifically bind to CD44 and/orEphA2 on the surface of cancer cells. The immunoliposomes can be loadedwith one or more of the anti-cancer agents, such as small molecule,peptide, and/or nucleic acid (e.g. siRNAs) or those set forth below inTable 5. Methods of making and loading lipidic nanoparticles, such asliposomes and immunoliposomes, are known in the art, e.g. US20100068255, US 20100008978, US 20090171077, US 20090155272, US20070116753, US 20070110798, US 20070031484, US 20060147513, US20050112065, US 20040037874, US 20040209366, US 20030003143, U.S. Pat.No. 7,135,177, U.S. Pat. No. 7,022,336, U.S. Pat. No. 6,803,053, U.S.Pat. No. 6,528,087, U.S. Pat. No. 6,214,388, U.S. Pat. No. 6,210,707,U.S. Pat. No. 6,110,491, U.S. Pat. No. 5,980,935, U.S. Pat. No.5,380,531, U.S. Pat. No. 7,507,407, U.S. Pat. No. 7,479,276, and U.S.Pat. No. 7,462,603.

The table below lists some examples of anti-cancer agents that may beused to modify the subject antibodies, e.g. by linking the agentcovalently or noncovalently to the antibody. As discussed later below,any agents listed below may also be formulated with the subjectantibodies in a composition or administered in a combination therapy inthe subject methods.

TABLE 5 Anti-Cancer Agents Anti-cancer agent Comments ExamplesAntibodies Antibodies which bind IGF-1R A12 (fully humanized mAb) (a)antibodies other than (insulin-like growth factor type 1 19D12 (fullyhumanized mAb) anti-EphA2 antibodies or receptor), which is expressed onCP751-871 (fully humanized anti-CD44 antibodies; and the cell surface ofmost human mAb) (b) anti-EphA2 antibodies cancers H7C10 (humanized mAb)or anti-CD44 antibodies alphaIR3 (mouse) antibodies which bind scFV/FC(mouse/human different epitopes of EphA2 chimera) or CD44 than theEM/164 (mouse) antibodies disclosed herein AMG 479 (fully humanized mAb;Amgen) IMCA 12 (fully humanized mAb; Imclone) NSC-742460 (Dyax) MR-0646,F50035 (Pierre Fabre Medicament, Merck) Antibodies which bind matuzumab(EMD72000) EGFR; Mutations affecting EGFR Erbitux ®/cetuximab (Imclone)expression or activity can result in Vectibix ®/panitumumab cancer(Amgen) mAb 806 nimotuzumab (TheraCIM) INCB7839 (Incyte) panitumumab(Vectibix ®; Amgen) Antibodies which bind AVEO (AV299) (AVEO) cMET(mesenchymal epithelial AMG102 (Amgen) transition factor); a member ofthe 5D5 (OA-5D5) (Genentech) MET family of receptor tyrosine kinases)Anti-ErbB3 antibodies 1B4C3; 2D1D12 (U3 Pharma AG) U3-1287/AMG888 (U3Pharma/Amgen) Anti-ErbB2 (HER2) antibodies MM-121 (MerrimackPharmaceuticals) Anti-ErbB2/anti-ErbB3 Herceptin ® (trastuzumab;bispecific antibodies Genentech/Roche) binds ectodomain Domain II ofErbB2; Omnitarg ® (pertuzumab; 2C4, R1273; Genentech/Roche) binds DomainIV of ErbB2 MM-111 (Merrimack Pharmaceuticals) Small Molecules TargetingIGF-1R (insulin-like growth factor NVP-AEW541-A IGF1R type 1 receptor),which is BMS-536,924 (1H- expressed on the cell surface ofbenzoimidazol-2-yl)-1H-pyridin- must human cancers 2-one) BMS-554,417Cycloligan TAE226 PQ401 Small Molecules Targeting EGFR; Mutationsaffecting EGFR Iressa ®/gefitinib (AstraZeneca) EGFR expression oractivity can result in CI-1033 (PD 183805) (Pfizer) cancerTYVERB/lapatinib (GlaxoSmithKline) Tykerb ®/lapatinib ditosylate(SmithKline Beecham) Tarceva ®/Erlotinib HCL (OSI Pharma) PKI-166(Novartis) PD-158780 EKB-569 Tyrphostin AG 1478(4-(3-Chloroanillino)-6,7- dimethoxyquinazoline) Small Molecules TargetingErbB2, also known as HER2, a HKI-272 (neratinib; Wyeth) ErbB2 member ofthe ErbB family of KOS-953 (tanespimycin; Kosan receptors, which isexpressed on Biosciences) certain cancer cells Small Molecules TargetingcMET (Mesenchymal epithelial PHA665752 cMET transition factor); a memberof the ARQ 197 (ArQule) MET family of receptor tyrosine ARQ-650RP(ArQule) kinases) Antimetabolites An antimetabolite is a chemicalflourouracil (5-FU) with a similar structure to a capecitabine/XELODA ®(HLR substance (a metabolite) required Roche) for normal biochemicalreactions, 5-trifluoromethyl-2′- yet different enough to interferedeoxyuridine with the normal functions of cells, methotrexate sodium(Trexall) including cell division. (Barr) raltitrexed/Tomudex ®(AstraZaneca) pemetrexed/Alimta ® (Lilly) tegafur cytosine arabinoside(Cytarabine, Ara-C)/tioguanine/ Lanvis ® (GlaxoSmithKline) 5-azacytidine6-mercaptopurine (Mercaptopurine, 6-MP) azathioprine/Azasan ® (AAIPHARMALLC) 6-thioguanine (6-TG)/ Purinethol ® (TEVA) pentostatin/Nipent ®(Hospira Inc.) fludarabine phosphate/ Fludara ® (Bayer Health Care)cladribine/Leustatin ® (2-CdA, 2- chlorodeoxyadenosine) (Ortho Biotech)floxuridine (5-fluoro-2′- deoxyuridine)/FUDR ® (Hospira, Inc,)Alkylating agents An alkylating antineoplastic agent RibonucleotideReductase is an alkylating agent that attaches Inhibitor (RNR) an alkylgroup to DNA. Since cyclophosphamide/Cytoxan ® cancer cells generallyproliferate (BMS)/ unrestrictively more than do Neosar ® (TEVA) healthycells they are more ifosfamide/Mitoxana ® (ASTA sensitive to DNA damage,and Medica) alkylating agents are used ThioTEPA (Bedford, Abraxis,clinically to treat a variety of Teva) tumors. BCNU→1,3-bis(2-chloroethyl)- 1-nitosourea CCNU→ 1,-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (methyl CCNU) hexamethylmelamine (altretamine,HMM)/Hexalen ® (MGI Pharma Inc.) busulfan/Myleran ® (GlaxoSmithKline)procarbazine HCL/Matulane ® (Sigma Tau) Dacarbazine (DTIC ®)chlorambucil/Leukaran ® (SmithKline Beecham) Melphalan/Alkeran ®(GlaxoSmithKline) cisplatin (Cisplatinum, CDDP)/ Platinol (BristolMyers) carboplatin/Paraplatin (BMS) oxaliplatin/Eloxitan ® (Sanofi-Aventis US) Bendamustine carboquone carmustine chloromethine dacarbazine(DTIC) fotemustine lomustine mannosulfan nedaplatin nimustineprednimustine ranimustine satraplatin semustine streptozocintemozolomide treosulfan triaziquone triethylene melamine triplatintetranitrate trofosfamide uramustine Topoisomerase inhibitorsTopoisomerase inhibitors are doxorubicin/Doxil ® (Alza) chemotherapyagents designed to daunorubicin citrate/ interfere with the action ofDaunoxome ® (Gilead) topoisomerase enzymes mitoxantrone/Novantrone(topoisomerase I and II), which are (EMD Serono) enzymes that controlthe changes actinomycin D in DNA structure by catalyzing theetoposide/Vepesid ® (BMS)/ breaking and rejoining of the Etopophos ®(Hospira, Bedford, phosphodiester backbone of DNA Teva Parenteral, Etc.)strands during the normal cell topotecan/Hycamtin ® cycle.(GlaxoSmithKline) teniposide (VM-26)/Vumon ® (BMS) irinotecan (CPT-11)/camptosar ® (Pharmacia & Upjohn) SN38 camptothecin (CPT) belotecanrubitecan Microtubule targeting Microtubules are one of thevincristine/Oncovin ® (Lilly) agents components of the cytoskeleton.vinblastine They have diameter of ~24 nm sulfate/Velban ®(discontinued)and length varying from several (Lilly) micrometers to possiblyvinorelbine tartrate/Navelbine ® millimeters in axons of nerve cells.(PierreFabre) Microtubules serve as structural vindesinesulphate/Eldisine ® components within cells and are (Lilly) involved inmany cellular paclitaxel/Taxol ® (BMS) processes including mitosis,docetaxel/Taxotere ® (Sanofi cytokinesis, and vesicular Aventis US)transport. Nanoparticle paclitaxel (ABI- 007)/Abraxane ® (AbraxisBioScience, Inc.) ixabepilone/IXEMPRA ™ (BMS) larotaxel ortataxeltesetaxel vinflunine colchicine Kinase inhibitors Tyrosine kinases areenzymes imatinib mesylate/Gleevec within the cell that function to(Novartis) attach phosphate groups to the sunitinib malate/Sutent ®amino acid tyrosine. By blocking (Pfizer) the ability of proteintyrosine sorafenib tosylate/Nexavar ® kinases to function, these (Bayer)compounds provide a tool for nilotinib hydrochloride controllingcancerous cell growth. monohydrate/Tasigna ® (Novartis) AMG 386 (Amgen)axitinib (AG-013736; Pfizer, Inc.) bosutinib (SKI-606; Wyeth) brivanibalalinate (BMS-582664; BMS) cediranib (AZD2171; Recentin, AstraZeneca)dasatinib (BMS-354825: Sprycel ®; BMS) lestaurtinib (CEP-701; Cephalon)motesanib diphosphage (AMG- 706; Amgen/Takeda) pazopanib HCL (GW786034;Armala, GSK) semaxanib (SU5416; Pharmacia) vandetanib (AZD647; Zactima;AstraZeneca) vatalanib (PTK-787; Novartis, Bayer Schering Pharma) XL184(NSC718781; Exelixis, GSK) Protein synthesis inhibitors Induces cellapoptosis L-asparaginase/Elspar ® (Merck & Co.) Immunotherapeutic agentsInduces cancer patients to exhibit Alpha interferon immuneresponsiveness Angiogenesis Inhibitor/ Avastin ® (Genentech) IL-2→Interleukin 2 (Aldesleukin)/Proleukin ® (Chiron) IL-12→ Interleukin 12Hormonal therapies Hormonal therapies associated Ttoremifenecitrate/Fareston ® with menopause and aging seek to (GTX, Inc.) increasethe amount of certain fulvestrant/Faslodex ® hormones in the body to(AstraZeneca) compensate for age- or disease- raloxifene HCL/Evista ®(Lilly) related hormonal declines. anastrazole/Arimidex ® Hormonaltherapy as a cancer (AstraZeneca) treatment generally either reducesletrozole/Femara ® (Novartis) the level of one or more specificfadrozole (CGS 16949A) hormones, blocks a hormone fromexemestane/Aromasin ® interacting with its cellular (Pharmacia & Upjohn)receptor or otherwise alters the leuprolide acetate/Eligard ® cancer'sability to be stimulated by (QTL USA) hormones to grow and spread.Lupron ® (TAP Pharm.) Such hormonal therapies thus goserelinacetate/Zoladex ® include hormone antagonists and (AstraZeneca) hormonesynthesis inhibitors. In triptorelin pamoate/Trelstar ® some instanceshormone agonists (Watson Labs) may also be used as anticancerbuserelin/Suprefact ® (Sanofi hormonal therapies. Aventis) nafarelincetrorelix/Cetrotide ® (EMD Serono) bicalutamide/Casodex ® (AstraZeneca)nilutamide/Nilandron ® (Aventis Pharm.) megestrol acetate/Megace ® (BMS)somatostatin Analogs (e.g., Octreotide acetate/ Sandostatin ®(Novartis)) abarelix (Plenaxis ™; Amgen) abiraterone acetate (CB7630;BTG plc) afimoxifene (TamoGel; Ascend Therapeutics, Inc.) aromataseinhibitor (Atamestane plus toremifene; Intarcia Therapeutics, Inc.)arzoxifene (Eli Lilly & Co) Asentar ™; DN-101 (Novacea; Oregon HealthSciences U) flutamide (Eulexin ®, Schering; Prostacur, LaboratoriosAlmirall, S.A) letrozole (CGS20267) (Femara ®, Chugai; Estrochek ®,(Jagsonpal Pharmaceuticals Ltd;) Delestrogen ®, estradiol valerate(Jagsonpal) magestrol acetate/Megace ® medroxyprogesteone acetate(Veraplex ®; Combiphar) MT206 (Medisyn Technologies, Inc.) nandrolonedecanoate (Zestabolin ®; Mankind Pharma Ltd) tamoxifen (Taxifen ®, YungShin Pharmaceutical; Tomifen ®, Alkem Laboratories Ltd.) tamoxifencitrate (Nolvadex, AstraZeneca; soltamox, EUSA Pharma Inc; tamoxifencitrate SOPHARMA, Sopharma JSCo.) Glucocorticoids Anti-inflammatorydrugs used to predinsolone reduce swelling that causes cancerdexamethasone/Decadron ® pain. (Wyeth) prednisone (Deltasone, Orasone,Liquid Pred, Sterapred ®) Aromatase inhibitors Includes imidazolesKetoconazole Exemestane (Aromasin ®) mTOR inhibitors The mTOR signalingpathway was sirolimus (Rapamycin)/ originally discovered duringRapamune ® (Wyeth) studies of the immunosuppressive Temsirolimus(CCI-779)/ agent rapamycin. This highly Torisel ® (Wyeth) conservedpathway regulates cell Deforolimus (AP23573) (Ariad proliferation andmetabolism in Pharm.) response to environmental factors, Everolimus(RAD001)/ linking cell growth factor receptor Certican ® (Novartis)signaling via phosphoinositide-3- kinase (PI-3K) to cell growth,proliferation, and angiogenesis. Chemotherapeutic agents adriamycin,5-fluorouracil, cytoxin, bleomycin, mitomycin C, daunomycin,carminomycin, aminopterin, dactinomycin, mitomycins, esperamicins,clofarabine, mercaptopurine, pentostatin, thioguanine, cytarabine,decitabine, floxuridine, gemcitabine (Gemzar), enocitabine, sapacitabineProtein Kinase B (PKB) AKT Inhibitor Astex ® (Astex InhibitorsTherapeutics) AKT Inhibitors NERVIANO (Nerviano Medical Sciences) AKTKinase Inhibitor TELIK (Telik Inc) AKT DECIPHERA (DecipheraPharmaceuticals, LLC) perifosine (KRX0401, D-21266; KeryxBiopharmaceuticals Inc, AEterna Zentaris Inc) perifosine with Docetaxel(Keryx Biopharmaceuticals Inc, AEterna Zentaris Inc) perifosine withGemcitabine (AEterna Zentaris Inc) perifosine with paclitaxel (AEternaZentaris Inc) protein kinase-B inhibitor DEVELOGEN (DeveloGen AG) PX316(Oncothyreon, Inc.) RX0183 (Rexahn Pharmaceuticals Inc) RX0201 (RexahnPharmaceuticals Inc) VQD002 (VioQuest Pharmaceuticals Inc) XL418(Exelixis Inc) ZEN027 (AEterna Zentaris Inc) Phosphatidylinositol 3-BEZ235 (Novartis AG) Kinase (PI3K) Inhibitors BGT226 (Novartis AG)CAL101 (Calistoga Pharmaceuticals, Inc.) CHR4432 (Chroma TherapeuticsLtd) Erk/PI3K Inhibitors ETERNA (AEterna Zentaris Inc) GDC0941(Genentech Inc/Piramed Limited/Roche Holdings Ltd) enzastaurin HCL(LY317615; Enzastaurin; Eli Lilly) LY294002/Wortmannin PI3K InhibitorsSEMAFORE (Semafore Pharmaceuticals) PX866 (Oncothyreon, Inc.) SF1126(Semafore Pharmaceuticals) VMD-8000 (VM Discovery, Inc.) XL147 (ExelixisInc) XL147 with XL647 (Exelixis Inc) XL765 (Exelixis Inc) PI-103(Roche/Piramed) Cyclin Dependent Kinase CYC200, R-roscovitine Inhibitors(Seliciclib; Cyclacel Pharma) NSC-649890, L86-8275, HMR- 1275(alvocidib; NCI) TLr9, CD289 IMOxine (Merck KGaA) HYB2055 (Idera)IMO-2055 (Isis Pharma) 1018 ISS (Dynavax Technologies/UCSF) PF-3512676(Pfizer) Enzyme Inhibitor lonafarnib(SCH66336; Sarasar; SuperGen, UArizona) Anti-TRAIL AMG-655 (Aeterna Zentaris, Keryx Biopharma)Apo2L/TRAIL, AMG951 (Genentech, Amgen) APOMAB (fully humanized mAb;Genentech) MEK Inhibitors Mitogen-Activated Protein Kinase ARRY162(Array BioPharma Kinase 1 (MAP2K1); Mitogen- Inc) Activated ProteinKinase Kinase 2 ARRY704 (Array BioPharma (MAP2K2) Inc) ARRY886 (ArrayBioPharma Inc) AS703026 (Merck Serono S.A) AZD6244 (AstraZeneca Plc)AZD8330 (AstraZeneca Plc) RDEA119 (Ardea Biosciences, Inc.) RDEA436(Ardea Biosciences, Inc.) XL518 (Exelixis Inc; Genentech Inc)Miscellaneous Inhibitors Imprime PGG (Biothera) CHR-2797(AminopeptidaseM1 inhibitor; Chroma Therapeutics) E7820, NSC 719239(Integrin- alpha2 inhibitor, Eisai) INCB007839 (ADAM 17, TACE Inhibitor;Incyte) CNF2024, BIIB021 (Hsp90 Inhibitor; Biogen Idec) MP470, HPK-56(Kit/Mel/Ret Inhibitor; Schering-Plough) SNDX-275/MS-275 (HDACInhibitor; Syndax) Zarnestra ™, Tipifarnib, R115777 (Ras Inhibitor;Janssen Pharma) volociximab; Eos 200-4, M200 (alpha581 integrininhibitor; Biogen Idec; Eli Lilly/UCSF/PDL BioPharma) apricoxib (TP2001;COX-2 Inhibitor, Daiichi Sankyo; Tragara Pharma)

The antibodies of the present disclosure can optionally be modified toprovide for improved pharmacokinetic profile (e.g., by PEGylation,hyperglycosylation, and the like). Modifications that can enhance serumhalf-life are of interest. A subject antibody may be “PEGylated”, ascontaining one or more poly(ethylene glycol) (PEG) moieties. Methods andreagents suitable for PEGylation of a protein are well known in the artand may be found in U.S. Pat. No. 5,849,860. PEG suitable forconjugation to a protein is generally soluble in water at roomtemperature, and has the general formula R(O—CH₂—CH₂)_(n)O—R, where R ishydrogen or a protective group such as an alkyl or an alkanol group, andwhere n is an integer from 1 to 1000. Where R is a protective group, itgenerally has from 1 to 8 carbons.

The PEG conjugated to the subject protein can be linear. The PEGconjugated to the subject protein may also be branched. Branched PEGderivatives such as those described in U.S. Pat. No. 5,643,575,“star-PEG's” and multi-armed PEG's such as those described in ShearwaterPolymers, Inc. catalog “Polyethylene Glycol Derivatives 1997-1998.” StarPEGs are described in the art including, e.g., in U.S. Pat. No.6,046,305.

Where the subject antibody is to be isolated from a source, the subjectprotein can be conjugated to moieties the facilitate purification, suchas members of specific binding pairs, e.g., biotin (member ofbiotin-avidin specific binding pair), a lectin, and the like. A subjectprotein can also be bound to (e.g., immobilized onto) a solid support,including, but not limited to, polystyrene plates or beads, magneticbeads, test strips, membranes, and the like.

Where the antibodies are to be detected in an assay, the subjectproteins may also contain a detectable label, e.g., a radioisotope(e.g., ¹²⁵I; ³⁵S, and the like), an enzyme which generates a detectableproduct (e.g., luciferase, β-galactosidase, horse radish peroxidase,alkaline phosphatase, and the like), a fluorescent protein, achromogenic protein, dye (e.g., fluorescein isothiocyanate, rhodamine,phycoerythrin, and the like); fluorescence emitting metals, e.g., ¹⁵²Eu,or others of the lanthanide series, attached to the protein throughmetal chelating groups such as EDTA; chemiluminescent compounds, e.g.,luminol, isoluminol, acridinium salts, and the like; bioluminescentcompounds, e.g., luciferin; fluorescent proteins; and the like. Indirectlabels include antibodies specific for a subject protein, wherein theantibody may be detected via a secondary antibody; and members ofspecific binding pairs, e.g., biotin-avidin, and the like.

Any of the above elements that are used to modify the subject antibodymay be linked to the antibody via a linker, e.g. a flexible linker. Ifpresent, the linker molecules are generally of sufficient length topermit the antibody and a linked carrier to allow some flexible movementbetween the antibody and the carrier. The linker molecules are generallyabout 6-50 atoms long. The linker molecules may also be, for example,aryl acetylene, ethylene glycol oligomers containing 2-10 monomer units,diamines, diacids, amino acids, or combinations thereof.

Where the linkers are peptide, the linkers can be of any of a suitableof different lengths, such as from 1 amino acid (e.g., Gly) to 20 ormore amino acids, from 2 amino acids to 15 amino acids, from 3 aminoacids to 12 amino acids, including 4 amino acids to 10 amino acids, 5amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 aminoacids to 8 amino acids, and may be 1, 2, 3, 4, 5, 6, or 7 amino acids.

Flexible linkers include glycine polymers (G)_(n), glycine-serinepolymers (including, for example, (GS)_(n), GSGGS_(n) (SEQ ID NO: 64)and GGGS_(n) (SEQ ID NO: 65), where n is an integer of at least one),glycine-alanine polymers, alanine-serine polymers, and other flexiblelinkers known in the art. Glycine and glycine-serine polymers may beused where relatively unstructured amino acids are of interest, and mayserve as a neutral tether between components. Examples of flexiblelinkers include, but are not limited GGSG (SEQ ID NO:66), GGSGG (SEQ IDNO:67), GSGSG (SEQ ID NO: 68), GSGGG (SEQ ID NO: 69), GGGSG (SEQ ID NO:70), GSSSG (SEQ ID NO: 71), and the like. The ordinarily skilled artisanwill recognize that design of a peptide conjugated to any elementsdescribed above can include linkers that are all or partially flexible,such that the linker can include a flexible linker as well as one ormore portions that confer less flexible structure.

Human Engineered Antibody

Where the antibodies of the present disclosure that binds CD44 or EphA2are not human, the antibodies can be humanized. As used herein, ahumanized antibody is a recombinant polypeptide that is derived from anon-human (e.g., rodent) antibody and has been modified to contain atleast a portion of the framework and/or constant regions of a humanantibody.

Humanized antibodies also encompass chimeric antibodies and CDR-graftedantibodies in which various regions may be derived from differentspecies. Chimeric antibodies may be antibodies that include a variableregion from any source linked to a human constant region. Thus, inchimeric antibodies, the variable region can be non-human, and theconstant region is human. CDR-grafted antibodies are antibodies thatinclude the CDRs from a non-human “donor” antibody linked to theframework region from a human “recipient” antibody. For example, anantibody of the present disclosure in a form of scFV may be linked to ahuman constant region (e.g. Fc region) to be made into a humanimmunoglobulin.

Fc Region

An antibody of the present disclosure that binds CD44 or EphA2 maycontain an Fc region. The Fc region may be any of the naturallyoccurring isoforms found in human or other animals (e.g. derived fromany classes or subclasses of immunoglobulins) and can optionally befurther modified to have altered function. For example, the Fc regionmay be modified in one or more amino acid residue position to haveincreased effector functions, such as initiating cell-mediatedcytotoxicity or activating complement activity (e.g. C1q binding orcomplement dependent cytotoxicity), downregulating cell-surfacereceptor, etc. Details of Fc variants that may be used as antibodies ofthe present disclosure may be found in, for example, U.S. Pat. No.7,416,727, U.S. Pat. No. 7,371,826, U.S. Pat. No. 7,335,742, U.S. Pat.No. 7,355,008, U.S. Pat. No. 7,521,542, and U.S. Pat. No. 7,632,497.

Compositions

The subject compositions provide antibodies and/or nucleic acid encodingthereof, in which the antibodies bind to and are internalized by cancercells. The compositions of the present disclosure find use in treating asubject (e.g., a human) containg cancer, and may be suitable fortreatment during any stage of the disease. Compositions containing one,two, or more different antibodies can be provided as a pharmaceuticalcomposition and administered to a mammal (e.g., to a human) in needthereof.

Compositions contemplated herein may contain one, two, three, or moredifferent antibodies of the present disclosure (and/or nucleic acidsencoding thereof). For example, the composition can contain one or moreof the following: F2-1A6, F2-1H9, E8H11, E8H7, E8G12, E8F11, E8C9, D6G9,D6D3, D1C5, D1D1, HB8, HC2, HC4, HE3, HF1, HH3, 2D6, D2-1A7, D2-1A9,D2-1B1, A3H9, A3G3, A3D10, A3D1, A3C8, 1A3, 1A5, 1A8, 1A12, 1B2, 1C2,1C7, 1D8, or 15H11. The composition may optionally further includeantibodies containing one or more CDRs from these antibodies, and/or oneor more antibodies containing mutants or derivatives of theseantibodies.

An example of a composition of the present disclosure may include any ofthe combinations described above or one or more of the antibodiesdisclosed in FIG. 7, 8, or 9, or FIGS. 44-47. Where the compositioncontains two or more antibodies, each antibody can be specific to thesame or different epitopes or to epitopes on different antigens. Forexample, the composition may contain at least one antibody specific forthe epitope of CD44 or EphA2 and another antibody specific for anothercell-surface antigen, such as EGFR. The composition may also containdual-specific, polyspecific antibodies, or nucleic acids encodingthereof.

The antibodies of the present disclosure can be used individually,and/or in combination with each other (e.g. to form bispecific orpolyspecific antibodies), and/or in combination with other knownanti-cancer agents (e.g. antibodies for cancer treatment). For example,a composition, such as a liposome, can comprise two or more antibodies,in which at least one of the antibodies is an antibody of the presentdisclosure. As described above, the liposome may contain one or moreantibodies that are different than the subject antibodies. Such liposomemay be dual-specific, polyspecific, etc, so that the liposome isspecific for an additional epitope (e.g. an epitope in EGFR or HER2) inaddition to the epitope of the subject antibody.

Combinations can be provided in a single formulation or can be providedas separate formulations in a kit, where the separate formulations maycontain a single antibody or two antibodies. Such separate formulationsof a kit may be combined prior to administration or administered byseparate injection.

A subject pharmaceutical composition can be provided in apharmaceutically acceptable excipient, which can be a solution such asan aqueous solution, e.g., a saline solution, or can be provided inpowder form. A subject composition may comprise other components, suchas pharmaceutical grades of mannitol, lactose, starch, magnesiumstearate, sodium saccharin, talcum, cellulose, glucose, sucrose,magnesium, carbonate, and the like. The compositions may containpharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions such as pH adjusting and bufferingagents, toxicity adjusting agents and the like, for example, sodiumacetate, sodium chloride, potassium chloride, calcium chloride, sodiumlactate and the like.

A subject antibody, e.g., in the form of a pharmaceutically acceptablesalt, can be formulated for oral, topical or parenteral administrationfor use in the methods described later below. In certain embodiments,e.g., where an antibody is administered as a liquid injectable, e.g.,suitable for intravenous injection) an antibody formulation may be asterile, non-pyrogenic aqueous solution comprising salts (e.g., toadjust tonicity) buffers, preservatives, amino acids, and otherpharmaceutically acceptable carriers and excipients, and may be providedas a ready-to-use dosage form, or as a reconstitutable storage-stablepowder or liquid composed of pharmaceutically acceptable carriers andexcipients. Formulation for convection enhanced delivery may be asdescribed in, e.g., US 20090208422.

Compositions of the present disclosure can include a therapeuticallyeffective amount of a subject antibody, as well as any other compatiblecomponents, as needed. By “therapeutically effective amount” is meantthat the administration of that amount to an individual, either in asingle dose, as part of a series of the same or different antibody orcompositions, is effective to reduce the proliferation and/or metastasesof a cancerous cell in a subject or to provide any other detectabletherapeutic benefit. Such therapeutically effective amount of anantibody and its impact on cell growth includes cooperative and/orsynergistic inhibition of cell growth in conjunction with one or moreother therapies (e.g., immunotherapy, chemotherapy, radiation therapyetc.). As noted below, the therapeutically effective amount can beadjusted in connection with dosing regimen and diagnostic analysis ofthe subject's condition (e.g., monitoring for the presence or absence ofa cell surface epitopes using an antibody specific for CD44 and/orEphA2) and the like.

Amount and Dosage

The exact dose will be ascertainable by one skilled in the art. Thedosage can depend on a variety of factors including the strength of theparticular compound employed, the condition of the subject, and the bodyweight of the subject, as well as the severity of the illness and thestage of the disease. The size of the dose will also be determined bythe existence, nature, and extent of any adverse side-effects that mightaccompany the administration of a particular compound. As known in theart, adjustments based on age, body weight, sex, diet, time ofadministration, drug interaction and severity of condition may benecessary and will be ascertainable with routine experimentation bythose skilled in the art. A therapeutically effective amount is also onein which the therapeutically beneficial effects outweigh any toxic ordetrimental effects of the antibody or antibody fragment.

The amount of composition administered to a subject, e.g., a human, inthe context of the present disclosure should be sufficient to effect aprophylactic or therapeutic response in the animal over a reasonabletime frame, and varies depending upon the goal of the administration,the health and physical condition of the individual to be treated, age,the degree of resolution desired, the formulation of the antibodycomposition, the treating clinician's assessment of the medicalsituation, and other relevant factors. Thus it is expected that theamount will fall in a relatively broad range, but can nevertheless beroutinely determined through various features of the subject such asnote above.

As an example, a non-limiting range for a therapeutically orprophylactically effective amount of a subject antibody is from about0.1 mg/kg to about 20 mg/kg, e.g., from about 1 mg/kg to about 10 mg/kg.

The concentration of an antibody in a pharmaceutical formulations canvary from less than about 0.1%, usually at or at least about 2% to asmuch as 20% to 50% or more by weight, and will be selected primarily byconsideration of fluid volumes, viscosities, etc., in accordance withthe particular mode of administration selected and the patient's needs.The resulting compositions may be in the form of a solution, suspension,tablet, pill, capsule, powder, gel, cream, lotion, ointment, aerosol orthe like.

Also, suitable doses and dosage regimens can be determined bycomparisons to anticancer or immunosuppressive agents that are known toaffect the desired growth inhibitory or immunosuppressive response. Suchdosages include dosages which result in the low dose inhibition of cellgrowth, without significant side effects. In proper doses and withsuitable administration of certain compounds, the compounds of thepresent disclosure can provide for a wide range of intracellulareffects, e.g., from partial inhibition to essentially completeinhibition of cell growth. Dosage treatment may be a single doseschedule or a multiple dose schedule (e.g., including ramp andmaintenance doses). As indicated below, a subject composition may beadministered in conjunction with other agents, and thus doses andregiments can vary in this context as well to suit the needs of thesubject.

Combination Therapy

Any of a wide variety of cancer therapies can be combined in acomposition with a subject antibody. For example, agents used inchemotherapeutic treatment or biological response modifier treatment maybe present in the pharmaceutical composition comprising the antibody,such as an immunoliposome. Certain agents that can be used incombination with the subject antibodies are provided in Table 5 aboveand/or briefly discussed below.

Chemotherapeutic agents are non-proteinaceous compounds that reduceproliferation of cancer cells, and encompass cytotoxic agents andcytostatic agents. Non-limiting examples of chemotherapeutic agentsinclude alkylating agents, nitrosoureas, antimetabolites, antitumorantibiotics, plant (e.g., vinca) alkaloids, nucleic acids, such asinhibitory nucleic acids (e.g. siRNA), and steroid hormones.

Antimetabolite agents include folic acid analogs, pyrimidine analogs,purine analogs, and adenosine deaminase inhibitors, for example.

Suitable natural products and their derivatives, (e.g., vinca alkaloids,antitumor antibiotics, enzymes, lymphokines, and epipodophyllotoxins)can be used as anti-cancer agents. For example, taxanes, such aspaclitaxel, as well as any active taxane derivative such as docetaxel,or a taxane pro-drug such as is2′-(2-(N,N′-diethylamino)propionyl)-paclitaxel,7-(2-(N,N′-diethylamino)propionyl)-paclitaxel,2′-(2-(N,N′-diethylamino)propionyl)-docetaxel or7-(2-(N,N′-diethylamino)propionyl)-docetaxel, are suitable.

Other anti-proliferative cytotoxic agents are navelbene, CPT-11(irinotecan), anastrazole, letrazole, capecitabine, reloxafine,cyclophosphamide, ifosamide, and droloxafine. Microtubule affectingagents that have antiproliferative activity are also suitable for use.Hormone modulators and steroids (including synthetic analogs) aresuitable for use.

Diagnostic Methods

The present disclosure provides a method of detecting a tumor associatedantigen (e.g. CD44 or EphA2) in a biological sample in a subject or in asample isolated from a subject. The methods are useful to bothdiagnostic and prognostic purposes. A subject method generally involvescontacting a sample containing a cell with an antibody of the presentdisclosure; and detecting binding of the antibody to a cell in thesample. The cell can be in vitro, where the cell is in a biologicalsample obtained from a subject suspected for having cancer cells, asubject undergoing cancer treatment, or a subject being tested forsusceptibility to treatment. The cell can be in vivo, e.g., the cell isin a subject suspected for having cancer cells, a subject undergoingtreatment, or a subject being tested for susceptibility to treatment.

Antibodies can be used to detect cells expressing CD44 and/or EphA2 in abiological sample of a subject having or suspected of having cancerouscells via immunodiagnostic techniques. Such diagnostics can be useful toidentify patients amenable to the therapies disclosed later below,and/or to monitor response to therapy.

Suitable immunodiagnostic techniques include, but are not necessarilylimited to, both in vitro and in vivo (imaging) methods. For example,anti-CD44 or anti-EphA2 antibodies can be detectably labeled,administered to a subject suspected of having a cancer characterized bycell surface expression of CD44 or EphA2, and bound detectably labeledantibody detected using imaging methods available in the art.

The phrase “in vivo imaging” as used herein refers to methods ofdetecting the presence of an antibody (e.g. detectably labeled 2D6) inwhole, live mammal. Optically detectable proteins such as fluorescentantibodies and luciferases-conjugated antibodies may be detected by invivo imaging. Methods for using luciferases for real-time imaging ofluciferase expression in live animals can be readily adapted for use inthe subject methods disclosed herein (e.g., Greer L F et al.,Luminescence 2002, 17: 43-74). In vivo imaging of fluorescent proteinsin live animals is described in, e.g., Hoffman, Cell Death andDifferentiation 2002, 9:786-789. In vivo imaging may be used to provide2-D as well as 3-D images of a mammal. Radiolabeled antibodies, forexample, may be administered to a subject and the subject imaged with agamma camera. Charge-coupled device cameras, CMOS, or 3D tomographersmay used to carry out in vivo imaging. For example, Burdette J E Journalof Mol. Endocrin., 40: 253-261, 2008, reviews utilizing computedtomography, magnetic resonance imaging, ultrasonography, positronemission tomography, single-photon emission computed tomography (SPECT),etc. The information from many in vivo imaging methods as thosedescribed above can provide information on cancer cells in the subject.

Where the methods are in vitro, the biological sample can be any samplein which a cancer cell may be present, including but not limited to,blood samples (including whole blood, serum, etc.), tissues, whole cells(e.g., intact cells), and tissue or cell extracts. For example, theassay can involve detection of CD44 and/or EphA2 on live cells or cellsin a histological tissue sample. Particularly, detection can be assessedon an extracellular surface of a living cell. For example, the tissuesample may be fixed (e.g., by formalin treatment) and may be providedembedded in a support (e.g., in paraffin) or frozen unfixed tissue.

Assays can take a wide variety of forms, such as competition, directreaction, or sandwich type assays. Examples of assays include Westernblots; agglutination tests; enzyme-labeled and mediated immunoassays,such as enzyme-linked immunosorbent assays (ELISAs); biotin/avidin typeassays; radioimmunoassays; immunoelectrophoresis; immunoprecipitation,and the like. The reactions generally include detctable labelsconjugated to the antibody. Labels include those that are fluorescent,chemiluminescent, radioactive, enzymatic and/or dye molecules, or othermethods for detecting the formation of a complex between antigen in thesample and the antibody or antibodies reacted therewith.

Where a solid support is used, the solid support is usually firstreacted with a solid phase component under suitable binding conditionssuch that the antibody is sufficiently immobilized to the support.Sometimes, immobilization to the support can be enhanced by firstcoupling the antibody to a protein with better binding properties, orthat provides for immobilization of the antibody on the support with outsignificant loss of antibody binding activity or specificity. Suitablecoupling proteins include, but are not limited to, macromolecules suchas serum albumins including bovine serum albumin (BSA), keyhole limpethemocyanin, immunoglobulin molecules, thyroglobulin, ovalbumin, andother proteins well known to those skilled in the art. Other moleculesthat can be used to bind antibodies to a support includepolysaccharides, polylactic acids, polyglycolic acids, polymeric aminoacids, amino acid copolymers, and the like, with the proviso that themolecule used to immobilize the antibody does not adversely impact theability of the antibody to specifically bind antigen. Such molecules andmethods of coupling these molecules to the antibodies, are well known tothose of ordinary skill in the art.

An ELISA method can be used, wherein the wells of a microtiter plate arecoated with a subject antibody. A biological sample containing orsuspected of containing CD44 and/or EphA2, is then added to the coatedwells. After a period of incubation sufficient to allow antibodybinding, the plate(s) can be washed to remove unbound moieties and adetectably labeled secondary binding molecule added. The secondarybinding molecule is allowed to react with any captured antigen, theplate washed and the presence or absence of the secondary bindingmolecule detected using methods well known in the art.

Where desired, the presence or absence of bound CD44 and/or EphA2 frombiological sample can be readily detected using a secondary bindercomprising an antibody directed against the antibody ligands. Forexample, a number of anti-bovine immunoglobulin (Ig) molecules are knownin the art which can be readily conjugated to a detectable enzyme label,such as horseradish peroxidase, alkaline phosphatase or urease, usingmethods known to those of skill in the art. An appropriate enzymesubstrate is then used to generate a detectable signal. In other relatedembodiments, competitive-type ELISA techniques can be practiced usingmethods known to those skilled in the art.

Assays can also be conducted in solution, such that the antibodies andthe antigens form complexes under precipitating conditions. Anantibody-coated particle can be contacted under suitable bindingconditions with a biological sample suspected of containing the targetantigen to provide for formation of particle-antibody-antigen complexaggregates which can be precipitated and separated from the sample usingwashing and/or centrifugation. The reaction mixture can be analyzed todetermine the presence or absence of antibody-antigen complexes usingany of a number of standard methods, such as those immunodiagnosticmethods described above.

Alternatively, assays for cellular uptake in live cells can be anotherdiagnostic technique to positively identify cancerous cells. Since thesubject antibodies are specifically internalized by cells expressingCD44 and/or EphA2, the cells can be allowed for internalization of theantibodies and any antibodies that are not internalized be washed away(e.g. acid wash). The internalized antibodies may be detected via itslabel as contained with the cells (e.g. FACS, spectrometer, radioisotopecounter, etc.). Internalizing antibodies may also be selected for asdescribed in U.S. Pat. No. 7,045,283.

The diagnostic assays described herein can be used to determine whethera subject has a cancer that is more or less amenable to therapy usingantibody-based therapy, as well as monitor the progress of treatment ina subject. It also may be used to assess the course of other combinationtherapies. Thus, the diagnostic assays can inform selection of therapyand treatment regimen by a clinician.

The above-described assay reagents, including the antibodies of thepresent disclosure, can be provided in kits, with suitable instructionsand other necessary reagents, in order to conduct immunoassays asdescribed above. The kit can also contain, depending on the particularimmunoassay used, suitable labels and other packaged reagents andmaterials (i.e. wash buffers and the like). Standard immunoassays, suchas those described above, can be conducted using these kits.

Therapeutic Methods

A subject antibody finds therapeutic use in a variety of cancers.Subjects having, suspected of having, or at risk of developing cancerare contemplated for therapy and diagnosis described herein.

By “treatment” is meant that at least an amelioration of the symptomsassociated with the condition afflicting the host is achieved, whereamelioration is used in a broad sense to refer to at least a reductionin the magnitude of a parameter, e.g. symptom, associated with thecondition being treated. As such, treatment also includes situationswhere the pathological condition, or at least symptoms associatedtherewith, are completely inhibited, e.g., prevented from happening, orstopped, e.g. terminated, such that the host no longer suffers from thecondition, or at least the symptoms that characterize the condition.Thus treatment includes: (i) prevention, that is, reducing the risk ofdevelopment of clinical symptoms, including causing the clinicalsymptoms not to develop, e.g., preventing disease progression to aharmful state; (ii) inhibition, that is, arresting the development orfurther development of clinical symptoms, e.g., mitigating or completelyinhibiting an active disease, e.g., so as to decrease tumor load, whichdecrease can include elimination of detectable cancerous cells (e.g.metastatic cancer cells); and/or (iii) relief, that is, causing theregression of clinical symptoms.

A variety of subjects are treatable according to the methods. Generallysuch subjects are “mammals” or “mammalian,” where these terms are usedbroadly to describe organisms which are within the class mammalia,including the orders carnivore (e.g., dogs and cats), rodentia (e.g.,mice, guinea pigs, and rats), and primates (e.g., humans, chimpanzees,and monkeys). In many embodiments, the subjects will be humans.

In a related embodiment, the subject being treated possesses cells thatexpress (e.g. overexpresses) a tumor associated antigen, CD44 and/orEphA2. The antigen is expressed on the cancer cell surface and is oftenpresent at a higher level than a corresponding non-cancerous cell. Thisaspect can be beneficial in the context of the methods of the presentdisclosure in that cells expressing or presenting CD44 and/or EphA2 canbe amenable to treatment with an antibody of the present disclosure. Theantibody can be administered to a subject, for example, where therapy isinitiated at a point where presence of the antigen is not detectable,and thus is not intended to be limiting. It is also possible to initiateantibody therapy prior to the first sign of disease symptoms, at thefirst sign of possible disease, or prior to or after diagnosis of adisease.

Prodrugs of the antibody composition of the present disclosure are alsocontemplated in the methods described herein. Such prodrugs are ingeneral functional derivatives of the compounds that are readilyconvertible in vivo into the required compounds. Thus, in the methods ofthe present disclosure, the term “administering” encompassesadministering the compound specifically disclosed or with a compoundwhich may not be specifically disclosed, but which converts to thespecified compound in vivo after administration to the subject in needthereof. Conventional procedures for the selection and preparation ofsuitable prodrug derivatives are described, e.g., in Wermuth, “DesigningProdrugs and Bioprecursors” in Wermuth, ed. The Practice of MedicinalChemistry, 2d Ed., pp. 561-586 (Academic Press 2003).

Cancer

The antibody compositions may be advantageously used in an anti-cancertherapy, particularly where the cancerous cells present EphA2 and/orCD44 on an extracellularly accessible cell surface. One example is acancer that presents an epitope bound by F2-1A6, F2-1H9, E8H11, E8H7,E8G12, E8F11, E8C9, D6G9, D6D3, D1C5, D1D1, HB8, HC2, HC4, HE3, HF1,HH3, 2D6, D2-1A7, D2-1A9, D2-1B1, A3H9, A3G3, A3D10, A3D1, A3C8, 1A3,1A5, 1A8, 1A12, 1B2, 1C2, 1C7, 1D8, or 15H11.

Antibody compositions described herein can be administered to a subject(e.g. a human patient) to reduce proliferation of cancerous cells, e.g.,to reduce tumor size, reduce cancer load, reduce metastasis, and/orimprove the clinical outcome in patients. For example, antibodycompositions can be used to disrupt the cell cycle of the cancer cell,and facilitate entry of the cell into apoptosis. The methods relating tocancer contemplated herein include, for example, use of antibody therapyalone or in combination with anti-cancer vaccine or therapy.

Cancers particularly amenable to antibody therapy can be identified bymethods similar to the diagnostic methods described above and othersknown in the art.

Types of Cancer

Where the anti-cancer therapy comprises administration of an antibodycomposition described previously, the anti-cancer therapy can beparticularly directed to cancerous cells expressing cell-surfaceaccessible and/or solvent-exposed epitopes bound by the subjectantibodies, including metastatic cancer.

Examples of cancers presenting epitopes of CD44 that can be treated bythe subject methods include cancers of the breast (e.g. basal cellbreast cancer), colon, prostate, pancreas, etc. as well as adenoma andhead and neck squamous cell carcinoma (HNSCC). Other cancers amenable totreatment can also be any cancer that is metastic and/or has metastaticpotential.

Examples of cancers presenting epitopes of EphA2 include but not limitedto cancer cells of epithelial origin. For example, the cancer cells canbe derived from breast (e.g. basal cell breast cancer), skin (e.g.melanoma), lung, prostate, colon, and/or ovary. Other cancers cells forwhich anti-EphA2 antibodies have affinity may be liver cancer,esophageal squamous cell carcinoma, epidermoid cancer, pancreaticcancer, glioblastoma, neuroblastomas, and/or other neural cancers, forexample.

It should be noted that while EphA2 or CD44 may be expressed at higherlevels on a cancer cell compared to a non-cancerous cell, this is not alimitation of the therapies disclosed herein.

Carcinomas that can be amenable to therapy by a method disclosed hereininclude, but are not limited to, esophageal carcinoma, hepatocellularcarcinoma, basal cell carcinoma (a form of skin cancer), squamous cellcarcinoma (various tissues), bladder carcinoma, including transitionalcell carcinoma (a malignant neoplasm of the bladder), bronchogeniccarcinoma, colon carcinoma, colorectal carcinoma, gastric carcinoma,lung carcinoma, including small cell carcinoma and non-small cellcarcinoma of the lung, adrenocortical carcinoma, thyroid carcinoma,pancreatic carcinoma, breast carcinoma, ovarian carcinoma, prostatecarcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinoma,cystadenocarcinoma, medullary carcinoma, renal cell carcinoma, ductalcarcinoma in situ or bile duct carcinoma, choriocarcinoma, seminoma,embryonal carcinoma, Wilm's tumor, cervical carcinoma, uterinecarcinoma, testicular carcinoma, osteogenic carcinoma, epithelialcarcinoma, and nasopharyngeal carcinoma.

Sarcomas that can be amenable to therapy by a method disclosed hereininclude, but are not limited to, fibrosarcoma, myxosarcoma, liposarcoma,chondrosarcoma, chordoma, osteogenic sarcoma, osteosarcoma,angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's sarcoma,leiomyosarcoma, rhabdomyosarcoma, and other soft tissue sarcomas.

Other solid tumors that can be amenable to therapy by a method disclosedherein include, but are not limited to, glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma,melanoma, neuroblastoma, and retinoblastoma.

Leukemias that can be amenable to therapy by a method disclosed hereininclude, but are not limited to, a) chronic myeloproliferative syndromes(neoplastic disorders of multipotential hematopoietic stem cells); b)acute myelogenous leukemias (neoplastic transformation of amultipotential hematopoietic stem cell or a hematopoietic cell ofrestricted lineage potential; c) chronic lymphocytic leukemias (CLL;clonal proliferation of immunologically immature and functionallyincompetent small lymphocytes), including B-cell CLL, T-cell CLLprolymphocytic leukemia, and hairy cell leukemia; and d) acutelymphoblastic leukemias (characterized by accumulation of lymphoblasts).Lymphomas that can be treated using a method include, but are notlimited to, B-cell lymphomas (e.g., Burkitt's lymphoma); Hodgkin'slymphoma; non-Hodgkin's lymphoma, and the like.

Other cancers that can be amenable to treatment according to the methodsdisclosed herein include atypical meningioma (brain), islet cellcarcinoma (pancreas), medullary carcinoma (thyroid), mesenchymoma(intestine), hepatocellular carcinoma (liver), hepatoblastoma (liver),clear cell carcinoma (kidney), and neurofibroma mediastinum.

Other examples of cancers that can be amenable to treatment using amethods disclosed herein include, but are not limited to, cancers ofepithelial and neuroectodermal origin. Examples of epithelial origininclude, but are not limited to, small cell lung cancer, cancers of thebreast, eye lens, colon, pancreas, kidney, liver, ovary, and bronchialepithelium. The methods of the present disclosure may be used to treatcancer cells known to overexpress CD44 and/or EphA2.

Examples of cancers of neuroectodermal origin include, but are notlimited to, Ewings sarcoma, spinal tumors, brain tumors, supratenbrialprimative neuroectodermal tumors of infancy, tubulocystic carcinoma,mucinous tubular and spindle cell carcinoma, renal tumors, mediastinumtumors, neurogliomas, neuroblastomas, and sarcomas in adolescents andyoung adults.

Combinations with Other Cancer Therapies

As noted above, another feature of the methods is that an antibody canbe administered to the subject in combination with one or more othertherapies. Such therapy may be combined in a composition or beconjugated to the subject antibodies. In addition to being physicallycombined with antibodies disclosed herein (e.g., as a conjugate or in aliposome or other lipidic nanoparticle), one or more anti-cancer agents,such as those listed in Table 5 above, may be administered inconjunction with, either simultaneously or before or after,administration of an antibody disclosed herein.

A therapy or treatment other than administration of antibody compositioncal be administered anywhere from simultaneously, to up to 5 hours ormore, e.g., 10 hours, 15 hours, 20 hours or more, prior to or afteradministration of a subject antibody. A subject antibody and othertherapeutic intervention are administered or applied sequentially, e.g.,where a subject antibody is administered before or after anothertherapeutic treatment. A subject antibody and other therapy areadministered simultaneously, e.g., where a subject antibody and a secondtherapy are administered at the same time, e.g., when the second therapyis a drug it can be administered along with a subject antibody as twoseparate formulations or combined into a single composition that isadministered to the subject. Regardless of whether administeredsequentially or simultaneously, as illustrated above, the treatments areconsidered to be administered together or in combination for purposes ofthe present disclosure.

Additional standard anti-cancer therapeutics that may or may not beadministered in conjunction with a subject antibody, include but notlimited to immunotherapy, chemotherapeutic agents and surgery (e.g., asthose described further below). In addition, therapeutic administrationof a subject antibody can also be post-therapeutic treatment of thesubject with an anti-cancer therapy, where the anti-cancer therapy canbe, for example, surgery, radiation therapy, administration ofchemotherapeutic agents, and the like. Antibodies other than thosedisclosed herein, particularly monoclonal antibodies that can providefor complement-mediated killing, and/or antibody-dependent cellularcytotoxicity-mediated killing, of a target cell may also be used.

For example, a subject antibody can be administered in combination withone or more chemotherapeutic agents (e.g., cyclophosphamide,doxorubicin, vincristine and prednisone (CHOP)), and/or in combinationwith radiation treatment and/or in combination with surgicalintervention (e.g., pre- or post-surgery to remove a tumor), radiationtherapy, bone marrow transplantation, biological response modifiertreatment, and certain combinations of the foregoing. Radiation therapyincludes, but is not limited to, X-rays or gamma rays that are deliveredfrom either an externally applied source such as a beam, or byimplantation of small radioactive sources.

Routes of Administration

In practicing the methods, routes of administration (path by which asubject antibody is brought into a subject) may vary, whererepresentative routes of administration for a subject antibody aredescribed in greater detail below. A subject antibody alone or incombinations described above can be administered systemically (e.g., byparenteral, intravenous, intramuscular, intrathecal, intraventricular,or subcutaneous administration) or locally (e.g., at a local tumor site,e.g., by intratumoral administration (e.g., into a solid tumor, into aninvolved lymph node in a lymphoma or leukemia, or by convection enhanceddelivery, e.g. into the brain, e.g., as disclosed in US 20090209937),administration into a blood vessel supplying a solid tumor, etc.), intoa body cavity or lumen, or into an organ. These different routes ofadministration may be carried out by injection or infusion.

Formulations suitable for parenteral administration include aqueous andnon-aqueous, isotonic sterile injection solutions, which can containanti-oxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.The formulations can be presented in unit-dose or multi-dose sealedcontainers, such as ampules and vials, and can be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid excipient, for example, water, for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions can be prepared from sterile powders, granules, and tabletsof the kind previously described. Methods for preparing parenterallyadministrable compositions will be known or apparent to those skilled inthe art and are described in more detail in such publications asRemington's Pharmaceutical Science, 15th ed., Mack Publishing Company,Easton, Pa. (1980).

Formulations suitable for oral administration can consist of (a) liquidsolutions, such as an effective amount of the compound dissolved indiluents, such as water, saline, or orange juice; (b) capsules, sachetsor tablets, each containing a predetermined amount of the activeingredient, as solids or granules; (c) suspensions in an appropriateliquid; and (d) suitable emulsions. Tablet forms can include one or moreof lactose, mannitol, corn starch, potato starch, microcrystallinecellulose, acacia, gelatin, colloidal silicon dioxide, croscarmellosesodium, talc, magnesium stearate, stearic acid, and other excipients,colorants, diluents, buffering agents, moistening agents, preservatives,flavoring agents, and pharmacologically compatible excipients. Lozengeforms can comprise the active ingredient in a flavor, usually sucroseand acacia or tragacanth, as well as pastilles comprising the activeingredient in an inert base, such as gelatin and glycerin, or sucroseand acacia, emulsions, gels, and the like containing, in addition to theactive ingredient, such excipients as are known in the art.

The formulations of the present disclosure can be made into aerosolformulations to be administered via inhalation. These aerosolformulations can be placed into pressurized acceptable propellants, suchas dichlorodifluoromethane, propane, nitrogen and the like. They mayalso be formulated as pharmaceuticals for non-pressured preparationssuch as for use in a nebulizer or an atomizer.

Formulations suitable for topical administration may be presented as astransdermal compositions or transdermal delivery devices (“patches”),creams, gels, pastes, or foams, containing, in addition to the activeingredient, such carriers as are known in the art to be appropriate.

Suppository formulations are also provided by mixing with a variety ofbases such as emulsifying bases or water-soluble bases. Formulationssuitable for vaginal administration may be presented as pessaries,tampons, creams, gels, pastes, foams.

Unit dosage forms for oral or rectal administration such as syrups,elixirs, and suspensions may be provided wherein each dosage unit, forexample, teaspoonful, tablespoonful, tablet or suppository, contains apredetermined amount of the composition containing the antibodycompositions. Similarly, unit dosage forms for injection or intravenousadministration may comprise the antibody in a composition as a solutionin sterile water, normal saline or another pharmaceutically acceptablecarrier.

Administration of the therapy can be repeated over a desired period,e.g., repeated over a period of about 1 day to about 5 days or onceevery several days, for example, about five days, over about 1 month,about 2 months, etc. It also can be administered prior, at the time of,or after other therapeutic interventions, such as surgical interventionto remove cancerous cells. The antibody can also be administered as partof a combination therapy, in which at least one of an immunotherapy, acancer chemotherapy or a radiation therapy is administered to thesubject (as described in greater detail above).

Screening Methods

The present disclosure also provides methods to screen for antibodiesspecific for antigens expressed on the cell surface (e.g. tumorassociated antigens), as well as for internalization into mammaliancells upon binding to cell surface antigen. Some such methods are wellknown, see, e.g., U.S. Pat. No. 6,794,128 and U.S. Pat. No. 7,045,283.In one embodiment, methods are disclosed for screening antibodylibraries (e.g., phage display libraries) for antibodies that bind toparticular antigen(s) and are internalized into cells upon antigenbinding. The antibody may be selected for its binding to a TAA (e.g.CD44 and/or EphA2) of interest and/or to a cancer cell, for example. Themethods comprise initial internalizing antibody selection by severaliterated rounds of library screening comprising selection forinternalization on at least one mammalian cell line. Next, thecollection of phage that express internalizing antibodies was selectedagainst one or more antigen(s) (e.g., yeast displayed antigen, e.g.,antigen known to be associated with a mammalian cell type) to isolatephage displaying antibodies against the desired antigen(s). The methodmay be executed according to the phage display, yeast display, andinternalization selection methods described in the examples below.

Briefly, non-immune human scFv phage library is optionally depleted withcontrol cells that do not express (e.g., do not appreciably express) theantigen(s) to be selected against to get rid of antibodies that may benonspecifically interacting with cancer cells. The optionally depletedlibrary is incubated with the live cancer cells of interest. The cancercells may be derived from a known source or an unknown source. Thecancer cells can also be derived exclusively from one cell line or onetumor or from a plurality of different cell lines or a plurality ofdifferent tumors. The process selects for antibodies that areinternalized into cells by allowing the cells to endocytose theantibodies and stripping the cells of surface bound antibodies beforeproceeding to recover the phage from the cells. During panning andselection, the internalized phage are recovered from cell lysates andamplified. Multiple successive selection rounds (e.g. two or more)ensure selection of phage displaying a polypeptide that acts as aspecific internalizing antibody for the cancer of interest.

Optionally before incubation with the yeast library, the selected phagemay be depleted with control yeasts (e.g. yeasts that are not expressingan antigen of interest and/or expressing an irrelevant protein). Thephage previously selected for cancer cell internalization are thenincubated with yeast displaying one or more TAAs of interest (e.g. anextracellular domain of EphA2). The yeast incubated with the phage forfurther selection can express a plurality of TAAs, each being adifferent peptide fragment of the same full-length protein and/or eachderived from different TAA. Where the yeast display a plurality of TAA,the antibodies specific for different TAA may be selected in parallel.The TAA may also encompass known tumor associated antigen and/or antigenwhose cancer association is yet to be verified (e.g. suspected of beingassociated with cancer). After incubation, the phage bound to yeastdisplaying TAA of interest is eluted. Multiple successive rounds (e.g.two or more) may be carried out against the yeast library.

The stringency of the selection against yeast library can increase overeach successive round (e.g. two or more). Many techniques well known inthe art may be employed to increase the specificity of the recoveredphage. Examples include increased wash times, increased detergentconcentrations, increased salt concentrations, and inclusion of knownmacromolecular inhibitors (e.g. BPTI, Ecotin, and/or previouslyidentified antibody inhibitors). Characterization of antibodies mayinclude ELISAs and inhibition assays. Details on the assays to beperformed in the method for selecting and isolating a polypeptide thatcan act as an anti-TAA agent are known in the art.

Compared to the use of phage to display antigens, the use of a simpleeukaryote such as yeast for antigen display can result in a greaterproportion of antigens being displayed in their proper conformation.

Kits and Systems

Also provided are kits and systems that find use in practicing themethods, as described above. For example, kits and systems may includeone or more of the compositions described herein, such as an anti-CD44and/or anti-EphA2 antibody (e.g. 2D6, D2-1A7, D2-1A9, D2-1B1, A3H9,A3G3, A3D10, A3D1, A3C8, 1A3, 1A5, 1A8, 1A12, 1B2, 1C2, 1C7, 1D8, or15H11; or F2-1A6, F2-1H9, E8H11, E8H7, E8G12, E8F11, E8C9, D6G9, D6D3,D1C5, D1D1, HB8, HC2, HC4, HE3, HF1, or HH3), a nucleic acid encodingthe same (especially a nucleic acid encoding a CDR of a heavy and/orlight chain of any subject antibodies described above), or a cellcontaining the same. Other optional components of the kit include:buffers, etc., for administering the subject antibody, and/or forperforming a diagnostic assay. The recombinant nucleic acids of the kitmay also have restrictions sites, multiple cloning sites, primer sites,etc to facilitate their ligation to constant regions of nucleic acids.The various components of the kit may be present in separate containersor certain compatible components may be precombined into a singlecontainer, as desired.

The kits and systems for practicing the methods may include one or morepharmaceutical formulations that include the antibody compositionsdescribed herein. As such, the kits may include a single pharmaceuticalcomposition present as one or more unit dosages. The kits may alsoinclude two or more separate pharmaceutical compositions.

In addition to the above components, the kits may further includeinstructions for practicing the methods. These instructions may bepresent in the kits in a variety of forms, one or more of which may bepresent in or on the kit. One form in which these instructions may bepresent is as printed information on a suitable medium or substrate,e.g., a piece or pieces of paper on which the information is printed, inor on the packaging of the kit, in a package insert, etc. Yet anothermeans would be a computer readable medium, e.g., diskette, CD, etc., onwhich the information has been recorded. Yet another means that may bepresent is a website address which may be used via the internet toaccess the information at a removed site. Any convenient means may bepresent in the kits.

A kit may be provided for use in treating a host suffering from acellular proliferative disease. This kit includes a pharmaceuticalcomposition comprising antibody specific for EphA2 or CD44, andinstructions for the effective use of the pharmaceutical composition ina method of treating a host suffering from a cancerous condition byinhibiting the growth of a cancer cell in a subject. Such instructionsmay include not only the appropriate handling properties, dosingregiment and method of administration, and the like, but can furtherinclude instructions to optionally screen the subject for a CD44- and/orEphA2-associated disease. This aspect can assist the practitioner of thekit in gauging the potential responsiveness of the subject to treatmentwith an antibody of the present disclosure, including timing andduration of treatment relative to the type and growth stage of thecancer. Thus in another embodiment, the kit may further include anantibody or other reagent for detecting an epitope of EphA2 or CD44 onan extracellularly accessible surface of a cancer cell. In anotherembodiment, the kit includes antibody that comprises a conjugate with adetectable label, such as a fluorophore.

The term “system” as employed herein refers to a collection ofantibodies described herein and one or more second therapeutic agents,present in single or disparate compositions that are brought togetherfor the purpose of practicing the methods. For example, separatelyobtained antibody specific to a TAA and chemotherapy dosage formsbrought together and coadministered to a subject are a system accordingto the present disclosure.

EXAMPLES

The following examples are offered to illustrate, but not to limit anyembodiments provided by the present disclosure.

The following methods and materials were used in the present example.

Methods and Materials

Cell Lines, Media, Antibodies and Full-Length cDNA Clones.

Breast cancer cell lines MCF7, T47D, MDAMB453, MDAMB231, human mammaryepithelial cell (HMEC), and SUM52PE were obtained from the ATCC andClontech (HMEC), or from collections developed in the laboratories ofDr. Steve Ethier (SUM52PE). The cell lines were cultured usingconditions described previously (Neve R M et al. (2006) Cancer Cell10:515-27). Yeast strain EBY100 was grown in YPD medium (CurrentProtocols in Molecular Biology, John Wiley and Sons, Chapter 13.1.2).EBY100 transfected with expression vector pYD2 (Razai A et al. (2005) JMol Biol 351:158-69) was selected on SD-CAA medium (Current Protocols,Chapter 13). The Aga2p antigen fusion was expressed on the yeast surfaceby induction in SG-CAA medium (identical to SD-CAA medium except theglucose is replaced by galactose) at 20° C. for 24-48 hr as describedpreviously (Feldhaus M J et al. (2003) Nat Biotechnol 21:163-70).Bacteria strain E. coli DH5α and TG1 were used for the preparation ofplasmid DNA and the expression of soluble scFv antibodies respectively.SV5 antibody was purified from hybridoma supernatant using Protein G anddirectly labeled with Alexa-488 or Alexa-647 using a kit provided by themanufacturer (Invitrogen; Carlsbad, Calif.). Biotin conjugated rabbitanti-fd bacteriaphage was purchased from Sigma and used to detect phageantibodies. Monoclonal antibody D7 against EphA2 ECD was purchased fromUpstate Biotech, polyclonal goat anti-EphA2 and recombinant mouse EphrinA1 with human Fc fusion protein from R&D Systems, anti-CD44 antibody forWestern Blotting from NeoMarkers, and monoclonal anti-CD44 recognizinglink domain from Abcam. The full-length cDNA of EphA2 and CD44 wasobtained from the ATCC.

The phage library used in the examples below contains non-immune humansingle-chain Fv antibodies (scFv). Briefly, the library was generated byfirst constructing a cDNA library from RNAs of human spleen cells andperipheral blood lymphocytes. Heavy chain and light chain repertoireswere joined to form the scFV gene repertoire. The single chain Fv (scFv)gene repertoire from a naïve phagemid antibody library was subclonedinto a true phage vector to create a multivalently displayed scFv phagelibrary. For details, see Sheets M D et al. (1998) Proc Natl Acad SciUSA 95:6157-62 and O'Connell D et al. (2002) J Mol Biol 321:49-56.

Antigen and Antigen Domains Displayed on the Yeast Surface.

Primers annealing to antigen cDNA and having a 25-mer overlappingsequence with pYD2/NcoI-NotI-digested vector were designed to amplifyantigen domains by PCR using Pfu polymerase. After gel purification, theamplified antigen fragment and NcoI-NotI digested pYD2 vector were usedto transform LiAc-treated EBY100 cells by gap repair (Gietz R D et al.(1991) Yeast 7:253-63; Orr-Weaver T L et al. (1983) Proc Natl Acad SciUSA 80:4417-21). The transformation mixes were cultured and subculturedin SD-CAA, and induced by culturing in SG-CAA medium for 24-48 hours at18° C. To validate antigen display, anti-EphA2 (R&D) and recombinantmouse Ephrin A1 (R&D) were analyzed for binding to yeast displayed EphA2ECD, and anti-CD44 antibody (Abcam) was analyzed for binding to CD44domain 1 by flow cytometry. Briefly, the induced yeast cells (10⁶ cells)with specific displayed antigen domains were incubated with monoclonalor polyclonal antibodies (1 μg/ml) for 1 h at 4° C., detected usinganti-goat PE conjugate for anti-EphA2, anti-human (Fc specific) forrEphrinA1-human Fc fusion protein, and anti-rabbit PE for anti-CD44respectively, and co-stained with SV5-Alexa-647.

Optimization of Elution Buffer for Phage Antibody Selection.

Different elution buffer including phosphate buffered saline, pH 7.4(PBS), 40 mM 2-mercaptoethylamine (2-MEA), 1 mM dithiolthreitol (DTT),100 mM triethylamine (TEA) and 100 mM Glycine/150 mM NaCl/0.1% BSA/0.5%Tween 20 were evaluated for their ability to elute bound phage form theyeast surface. The elution time was 1 hour at 37° C. for PBS, 2-MEA andDTT, and 2 minutes at RT for TEA and glycine. After neutralizing with 10mM cysteine for 2-MEA and DTT elution, and ½ volume of 1M Tris-HCl (pH7.4) for TEA and glycine elutions, the eluted mixture was used to infectexponentially growing E. coli TG1 cells, and the titer of phagedetermined by serial dilution and plating on tetracycline resistantmedia.

Selection of Phage Antibodies Specific to Yeast Displayed AntigenDomains.

Human mammary epithelial cell (HMEC), luminal breast cancer cell lineSUM52PE, T47D, and MDAMB453 were used to deplete the phage library ofnonspecific binders by incubating 10¹² phage particles (Sheets M D etal. (1998) Proc Natl Acad Sci USA 95:6157-62; Huie M A et al. (2001)Proc Natl Acad Sci USA 98: 2682-7) with 10⁸ cells for 4 h at 4° C. Thedepleted phage library was then incubated with 5×10⁶ basal breast cancercell line MDAMB231 cells for 1 h at 4° C., followed by washing with coldPBS and incubation with 37° C.-prewarmed medium/10% FBS for 30 mM at 37°C. to enable the receptor mediated endocytosis of phage particles. Thecell surface was stripped by three incubations of five minutes with 4 mlof glycine buffer (150 mM NaCl, 0.1 M glycine, pH 2.5). The cells werethen trypsinized, washed with PBS, lysed with 1 ml of 100 mM TEA forfour minutes at 4° C. and neutralized with 0.5 ml of 1M Tris (pH 7.4).Internalized phage (TEA lysate) was amplified for further selections.

After two rounds of selection on MDAMB231 cells, the polyclonal phageantibodies were used to select phage antibodies specific to yeastdisplayed antigens EphA2 (Y-EphA2) and CD44 link domain (Y-CD44 D1). Theinduced yeast cells displaying an irrelevant protein were used todeplete the non-specific binders by incubating 2.5×10¹¹ phage particleswith 10⁹ yeast cells for 2 h at 4° C. The filtered supernatantcontaining the depleted phage library was then incubated with 2×10⁷yeast-cells displaying specific antigen domain for 1 h at 4° C. Yeastcells were washed with cold PBS ten times and pelleted bycentrifugation. The bound phage antibodies were eluted by incubatingyeast cells with 1 ml of 100 mM Glycine/150 mM NaCl/0.1% BSA/0.5% Tween20, neutralized with 0.5 ml of 1M Tris-HCl (pH 7.4), and amplified foranother round of selection. In the second round of selection, 2×10⁷yeast cells were used for both antigens, while 2.1×10¹² phage particlesfrom the first round selection were used for CD44 domain 1 compared to3.4×10¹¹ used for EphA2. Two rounds of selection were performed.

Characterization of Phage Antibodies.

After two rounds of selection, individual phage antibodies were preparedby growing single colonies in 96-well microtiter plates as described(O'Connell D et al. (2002) J Mol Biol 321:49-56). Binding of each phageantibody to yeast displayed antigen was determined by incubation of 10⁵yeast cells with 100 μl phage supernatant diluted in FACS buffer (PBSwith 1 mM MgCl₂, 0.1 mM CaCl₂ and 0.3% BSA) for 2 h at 4° C. in conical96-well microtiter plates, followed by incubation with biotinylatedanti-fd antibody and streptavidin-phycoerythrin conjugate (PE)(Jackson), and analyzed using a FACS LSRII (Becton Dickinson). Thenumber of unique phage antibodies was determined by patterns of BstNIdigestion of 18 scFv genes amplified by PCR from phage-infected bacteria(Marks, J D et al. (1991) J Mol Biol 222:581-97) and confirmed by DNAsequencing.

For binding to breast cancer cells and Ephrin A1 competitionexperiments, 5×10⁴ MDAMB231 cells were incubated with 10⁸ phageantibodies in the presence of recombinant mouse Ephrin A1 (R&D) atconcentration of 0 to 1000 ng/ml for 2 h at 4° C. The bound phageantibodies were detected by incubating cells with biotin conjugatedanti-fd antibody (1 μg/ml) (Sigma) for 30 min at 4° C. andstreptavidin-PE (Jackson) followed by flow cytometry analysis.

Immunoprecipitation and Western Blot Using scFv Antibodies.

MDAMB231 cell extracts were prepared using 1 ml of lysis buffer per T75culture flask, containing 0.5% NP40, 50 mM Tris (pH 7.4), 150 mM NaCl, 2mM DTT, and protease inhibitor cocktail (Sigma). Soluble scFv antibodieswith a (His)₆ tag at the C-terminal were generated by subcloning scFvgenes from the phage vector into the expression vector pUC119mycHis(Schier R et al. (1995) Immunotechnology 1:73-81), followed bypurification from the periplasmic fraction of E. coli TG1 by IMAC⁵¹using a Ni-NTA column (Qiagen) and gel filtration (Schier R et al.(1996) J Mol Biol 255:28-43). Cell extracts were incubated with scFv at26 μg/ml for 2 h at 4° C. before the immune complexes were captured onNi-NTA agarose. The agarose captured immune complexes were then washed 5times in lysis buffer and heated to 94° C. for 4 min in non-reducingprotein loading buffer. Immunoprecipitates were resolved by SDS-PAGE andanalyzed by Western Blotting using anti-EphA2 (Upstate) and anti-CD44(NeoMarkers) antibodies.

Immunofluorescence.

MDAMB231 cells were grown on coverslips to 70% of confluence in 12well-plates and incubated with 10¹¹ phage antibodies for three hours at37° C. The coverslips were washed once with PBS, three times for fiveminutes with glycine buffer (50 mM glycine (pH 2.5), 150 mM NaCl),neutralized with PEM (80 mM Potassium PIPES (pH 6.8), 5 mM EGTA (pH 7),2 mM MgCl₂), and fixed with PEM containing 4% (W/V) paraformaldehyde for30 min on ice. Cells were quenched with 0.1 M NH₄Cl, permeabilized with0.5% Triton X-100, and blocked with 5% non-fat dry milk in TBS-T bufferovernight at 4° C. After blocking endogenous biotin with Avidin-BiotinKit (Lab Vision), intracellular phages were detected with biotinylatedanti-fd polyclonal antibody (Sigma) and streptavidin Texas Red(Amersham). Coverslips were inverted on a slide on mounting medium andmicroscopic images were taken with a Zeiss LSM 510 laser scanningmicroscope (Zeiss, Germany).

Example 1 Display of Tumor Associated Antigens on the Surface of Yeast

Two TAA (CD44 and EphA2) are overexpressed in basal breast cancers(Hamilton S R et al. (2007) J Biol Chem 282:16667-80) and were selectedfor display on the surface of Sachromyces cerevisiae. For yeast surfacedisplay, the full-length extracellular domain (ECD) of EphA2 (aa 1-510)and the link domain of CD44 (aa 1-149) (domain 1) were cloned into theyeast display vector pYD2 for (C-terminal) fusion to Aga2 (Razai A etal. (2005) J Mol Biol 351:158-69). Vector DNA was used to transformEBY100, and cell surface display was induced. Both extracellular domainsof CD44 and EphA2 were well displayed on the yeast surface asquantitated by a monoclonal antibody to a C-terminal epitope tag (FIG.1). Specific binding to yeast displayed EphA2 and CD44 extracellulardomains of the EphA2 natural ligand Ephrin A1, and antibodies to EphA2and CD44 suggests that the domains are not only displayed but displayedin a form that can bind to ligand and can be specifically recognized byantibodies (FIG. 1).

Example 2 Efficient Recovery of Antigen Specific Phage Antibodies fromYeast Cell Surface Displayed Antigen

A scFv phage antibody that specifically bound to EphA2 was used to studythe ability to select phage antibodies on yeast displayed antigen.Approximately 10¹¹ phage particles displaying anti-EphA2 human scFv 2D6were incubated with 10⁸ yeast cells displaying the target antigen EphA2ECD (Y-EphA2). As a control, an identical number of anti-EphA2 phageantibodies were incubated with 10⁸ yeast cells displaying an irrelevantscFv (Y-CON). The recovery of anti-EphA2 phage antibody from yeastdisplaying the EphA2 ECD was more than 10⁴ fold higher than the recoveryof anti-EphA2 phage antibody from yeast displaying the scFv (FIG. 2,panel A). To determine the optimal buffer to elute phage antibodies fromthe yeast surface, different buffers (PBS, 2-MEA, DTT, triethylamine(TEA) and glycine) were evaluated. Although yeast surface display of TAAresults from the disulfide linkage between the Aga2 and Aga1 proteins onthe yeast surface, reducing agents, including 2-MEA and DTT, resulted inpoorer recovery of viable phage antibodies than spontaneous dissociationof phage by incubation in PBS (FIG. 2, panel B). In contrast, elutionwith high pH TEA buffer or low pH glycine buffer increased the number ofviable phage recovered approximately two fold, with low pH glycine beingthe optimal elution buffer of those studied. This elution buffer wasused for subsequent studies.

To determine the minimum frequency of a specific antibody within alibrary that can be enriched and selected, phage displayed anti-EphA2antibodies were serially diluted from 10⁹ to 10⁰ cfu and then mixed with10⁹ helper phage VCSM13. Phage mixtures were incubated with 10⁷ yeastcells displaying the EphA2 ECD or with yeast displaying the CD44 domain1, followed by washing, elution and titration of the recovered phages.With an input of 10² specific phage particles, about 12 phage wererecovered from yeast cells displaying EphA2, while an input of at least10⁵ phage were required before phage were present in the output whenselected on CD44 (FIG. 2, panel C). The average recovery of phageantibodies was 6.5×10⁻² for the specific antigen-antibody pair, and6.5×10⁻⁵ for the mismatched pair (Table 6), respectively. This highrecovery ratio for specific compared to non-specific phage suggestedthat it would be possible to enrich and select phage antibodies on yeastdisplayed antigen.

TABLE 6 Recovery of specific phage antibody from yeast displayedantigens. Ag:Ab PhAb Output/Input Y-EphA2 ECD:PhAb-EphA2 6 × 10⁻² Y-CD44D1:PhAb-CD44 7 × 10⁻² Y-EphA2 ECD:PhAb-CD44 3 × 10⁻⁵ Y-CD44D1:PhAb-EphA2 1 × 10⁻⁶ The indicated yeast displayed antigen wasincubated with 10⁹ phage and the titer of bound phage determined.Results are expressed as the ratio of output/input phage. Y = yeastdisplayed antigen; PhAb = phage displayed antibody.

Example 3 Selection of Antigen Specific Phage Antibodies on Yeast CellsDisplaying Tumor Antigens

The strategy used to select internalizing phage antibodies to specifictumor antigens is shown in FIG. 3. To ensure that phage antibodies boundtumor antigens as presented on the surface of human tumor cells andcould be internalized upon antigen binding, a starting phage library wasused. The polyclonal phage output after the second round of selection ofa non-immune human scFv phage library (O'Connell D et al. (2002) J MolBiol 321:49-56) was selected for endocytosis into MDAMB231 tumor cells.Based on the fact that basal subtype breast cancer cells over-expressEphA2 (Neve R M et al. (2006) Cancer Cell 10:515-27) and CD44 (HamiltonS R et al. (2007) J Biol Chem 282:16667-80), the phage output ofselection on the basal subtype breast cancer cell line MDAMB231 wasselected independently on yeast displayed EphA2 ECD (aa 25-534) and CD44domain 1 (aa 21-169). To remove phage antibodies that bound toirrelevant proteins on the yeast surface, 2.5×10¹¹ phage from the secondround output of the MDAMB231 selection were incubated first with 1×10⁹yeast cells displaying the scFv 4E17 (Y-CON). Then, the depleted phagelibrary was incubated with 2×10⁷ yeast cells displaying the relevantantigen (Y-Ag) (FIG. 3, panel A).

For both EphA2 ECD and CD44 domain 1 selections, the number of phagerecovered from each yeast cell in the second round of selectionincreased over 40 fold compared to the first round (Table 7), suggestingenrichment for phage binding yeast displayed tumor antigens. This wasverified by using polyclonal phage to stain the yeast displayedantigens. From both the first and second round of selections, polyclonalphage antibodies showed specific binding to the antigen domain that wasused for selection, with stronger staining after the second round ofselection (FIG. 3, panel B). In contrast, prior to selection, the inputphage antibody library gave no signal above background on yeast cellsdisplaying either EphA2 or CD44. Binding was specific for the yeastdisplayed antigen, since the polyclonal phage did not bind yeast cellsdisplaying an irrelevant protein (the scFv 4E17) (FIG. 3, panel B). Todetermine the frequency of binding phage antibodies, 96 individualclones were picked, phage produced, and the phage analyzed for bindingto the yeast displayed tumor antigen. After one round and two rounds ofselection, 31/96 (32.2%) and 39/96 (40.6%) of the clones from EphA2selection bound yeast cells displaying EphA2 ECD, and 11/96 (11.7%) and21/96 (21.9%) of the clones from CD44 selection bound yeast displayedCD44, respectively (FIG. 3, panel C)

TABLE 7 Phage display scFv antibody selection on yeast displayedantigens. Output Phage Antigen Input (cfu) Output (cfu) Input YeastRound 1 selection EphA2-ECD 2.5 × 10¹¹ 3.6 × 10⁶ 1.3 × 10⁻⁵ 0.18CD44-ECD D1 2.5 × 10¹¹ 9.8 × 10⁶ 3.9 × 10⁻⁵ 0.49 Round 2 selectionEphA2-ECD 3.4 × 10¹¹ 2.4 × 10⁸   7 × 10⁻⁴ 12 CD44-ECD D1 2.1 × 10¹² 4.3× 10⁸   2 × 10⁻⁴ 20 Phage input and output ratios during the first andsecond rounds of selection on yeast displayed antigens.

Example 4 Identification and Characterization of Phage Antibodies

Individual phage antibodies from the second round of selection thatbound the yeast displayed tumor antigen were analyzed by PCRfingerprinting and DNA sequencing of the scFv genes. The results areshown in FIG. 9, where the nucleic acid and amino acid sequences foreach of the isolated clones are shown. The sense strand is shown as thetop strand of the nucleic acid sequences in FIG. 9. The CDRs andframework regions are identified based on the Kabat database for eachantibody as shown in FIGS. 7 and 8.

Three unique human scFv antibodies (D2-1A7, D2-1A9 and D2-1B1) wereidentified which bound to EphA2, and two unique scFv (F2-1A6 and F2-1H9)were identified which bound to CD44 ECD domain 1. Characterization ofeach of these scFv on yeast displayed EphA2-ECD (Y-EphA2 ECD), CD44 ECDdomain 1 (Y-CD44 ECD D1) and full-length ECD (Y-CD44 ECD), and scFv 4E17(Y-CON) indicated that each scFv was specific for its target antigen(FIG. 4, panel A). Each unique phage antibody was also analyzed for itsability to bind the original selecting tumor cell line MDAMB231 by flowcytometry. Each phage antibody strongly stained MDAMB231 cells (FIG. 4,panel B). Since the initial selection of the phage antibody libraryaimed to target cell surface receptors specific to basal subtype breastcancer cells, the binding of the selected mAbs to basal and luminalbreast cancer cell lines was determined. Each mAb was relativelyspecific for basal breast cancer cell lines compared to luminal breastcancer cell lines (FIG. 4, panel C).

Example 5 Binding Specificity of Phage Antibodies

The binding of the identified EphA2 and CD44 antibodies to nativeantigens was also confirmed by immunoprecipitation of the receptors fromcell extracts of MDAMB231 cells followed by Western blotting with murinemonoclonal antibodies specific to EphA2 and CD44 (FIG. 5, panel A). Thespecificity of the EphA2 mAbs was further studied. The ability of eachof the EphA2 mAbs was evaluated for its ability to compete with thenatural ligand, ephrin A1 for binding to EphA2 on the surface ofMDAMB231 cells. Although the IC50 of ephrin A1 for phage antibodiesD2-1A7 and D2-1A9 at the given concentration differed by 9-fold, 1 μg/mlephrin A1 can fully block the cell binding of both D2-1A7 and D2-1A9phage antibodies (FIG. 5, panel B), indicating that these two antibodiesbind epitopes which overlap with Ephrin A1.

Example 6 Phage Antibodies are Internalized by MDAMB231 Cells

Since the phage antibodies identified by yeast display antigenbiopanning were originally selected for the ability to be endocytosedinto MDAMB231 cells (FIG. 3, panel A), it was anticipated that theywould be internalized efficiently. To determine whether D2-1A7, D2-1A9and F2-1A6 phage antibodies were endocytosed, the phage antibodies wereincubated with MDAMB231 cells at 37° C. to allow receptor mediatedendocytosis, the surface bound phage removed by stripping with low pHbuffer, and the internalized phage stained with anti-fd antibody andobserved using confocal microscopy (FIG. 6). Both D2-1A7 and D2-1A9anti-EphA2 antibodies gave strong intracellular staining, a controlphage gave no staining, and F2-1A6 anti-CD44 antibody gave a differentstaining pattern than the anti-EphA2 phage.

Example 7 Fd Phage and Phagemid Antibodies Specific for CD440R EphA2

To select antigen specific antibodies without the use of mammaliancells, the non-immune human scFv phage library (Sheets, 1998; Huie,2001) was incubated with yeast-displayed tumor associated antigens(TAAs) without prior selection on cancer cells. Specifically, 10¹²fd-phage particles (Huie et al. (2001) supra) or 10¹³ phagemid-phageparticles (Sheets et al (1998) supra) were first incubated with 10¹⁰yeast cells displaying an irrelevant protein for 2 h at 4° C. to removephage antibodies binding common yeast proteins. The depleted phagelibrary was then incubated with 10⁸ yeast cells displaying a specificantigen domain for 2 h at 4° C. (for example EphA2 or CD44). Yeast cellswere washed with cold PBS ten times and pelleted by centrifugation. Thebound phage antibodies were eluted by incubating yeast cells with 1 mlof 100 mM Glycine/150 mM NaCl/0.1% BSA/0.5% Tween 20, neutralized with0.5 ml of 1M Tris-HCl (pH 7.4), and amplified for another round ofselection. In the second round of selection, 2×10⁷ yeast cells were usedfor both antigens, while phage particles from the first round selectionwere used as the input phage library with 10¹¹ phage for fd library and10¹² for phagemid library respectively. Two rounds of selection wereperformed to enrich phage antibodies specific to the TAA used forselection. If two rounds of selection didn't enrich TAA specificantibodies, a third round of selection was performed, following the sameprotocol as the second round of selection.

The method for screening and characterization of monoclonal phageantibodies was the same as that described in Examples 4 and 5.

Five fd phage antibodies, designated E8H11, E8H7, E8G12, E8F11, andE8C9, were raised to CD44 link domain and were specific for CD44 linkdomain. Two fd phage antibodies, designated D6G9 and D6D3, were raisedto the CD44s (standard form), and were specific for the CD44s.

Two phagemid antibodies, designated D1C5 and D1D1, were raised to theCD44 link domain, and were specific for the CD44 link domain. Sixphagemid antibodies, designated HB8, HC2, HC4, HE3, HF1, and HH3, wereraised to CD44s and were specific for CD44s.

Five fd phage antibodies, designated A3H9, A3G3, A3D10, A3D1, and A3Cwere raised to EphA2 and were specific for EphA2. Eight phagemidantibodies, designated 1A3, 1A5, 1A8, 1A12, 1B2, 1C2, 1C7, and 1D8, wereraised to EphA2 and were specific for EphA2.

Another fd phage antibody, designated 15E11, was raised to EphA2 and wasspecific to EphA2.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes. While the subject antibody, method, andcomposition have been particularly shown and described with referencesto preferred embodiments thereof, it will be understood by those skilledin the art that various changes in form and details may be made thereinwithout departing from the scope of the invention encompassed by theappended claims.

1-43. (canceled)
 44. A monoclonal antibody that specifically binds anepitope of EphA2 that is specifically bound by antibody 2D6, D2-1A7,D2-1A9 or D2-1B1, or that competes with antibody 2D6, D2-1A7, D2-1A9 orD2-1B1, for binding to EphA2.
 45. The monoclonal antibody of claim 44,wherein said antibody comprises: a) a V_(H) CDR1, a V_(H) CDR2 and aV_(H) CDR3 of 2D6; b) a V_(H) CDR1, a V_(H) CDR2 and a V_(H) CDR3 ofD2-1A7; c) a V_(H) CDR1, a V_(H) CDR2 and a V_(H) CDR3 of D2-1A9; or d)a V_(H) CDR1, a V_(H) CDR2 and a V_(H) CDR3 of D2-1B1.
 46. Themonoclonal antibody of claim 44, wherein said antibody comprises: a) aV_(L) CDR1, a V_(L) CDR2 and a V_(L) CDR3 of 2D6; b) a V_(L) CDR1, aV_(L) CDR2 and a V_(L) CDR3 of D2-1A7; c) a V_(L) CDR1, a V_(L) CDR2 anda V_(L) CDR3 of D2-1A9; or d) a V_(L) CDR1, a V_(L) CDR2 and a V_(L)CDR3 of D2-1B1;
 47. A monoclonal antibody that specifically binds anepitope of EphA2 that is specifically bound by an antibody selected fromA3H9, A3G3, A3D10, A3D1, A3C8, 1A3, 1A5, 1A8, 1A12, 1B2, 1C2, 1C7, 1D8,and 15H11 or that competes with an antibody selected from A3H9, A3G3,A3D10, A3D1, A3C8, 1A3, 1A5, 1A8, 1A12, 1B2, 1C2, 1C7, 1D8, and 15H11for binding to EphA2.
 48. A monoclonal antibody that specifically bindsan epitope of CD44 that is specifically bound by antibody F2-1A6 orF2-1H9, or that competes with antibody F2-1A6 or F2-1H9, for binding toCD44.
 49. A monoclonal antibody that specifically binds an epitope ofCD44 that is specifically bound by an antibody selected from E8H11,E8H7, E8G12, E8F11, E8C9, D6G9, D6D3, D1C5, D1D1, HB8, HC2, HC4, HE3,HF1, and HH3; or that competes with an antibody selected from E8H11,E8H7, E8G12, E8F11, E8C9, D6G9, D6D3, D1C5, D1D1, HB8, HC2, HC4, HE3,HF1, and HH3 for binding to CD44.
 50. The monoclonal antibody of claim49, wherein said antibody comprises: a) a V_(H) CDR1, a V_(H) CDR2 and aV_(H) CDR3 of E8H11; b) a V_(H) CDR1, a V_(H) CDR2 and a V_(H) CDR3 ofE8H7; c) a V_(H) CDR1, a V_(H) CDR2 and a V_(H) CDR3 of E8G12; d) aV_(H) CDR1, a V_(H) CDR2 and a V_(H) CDR3 of E8F11; e) a V_(H) CDR1, aV_(H) CDR2 and a V_(H) CDR3 of E8C9; f) a V_(H) CDR1, a V_(H) CDR2 and aV_(H) CDR3 of D6G9; g) a V_(H) CDR1, a V_(H) CDR2 and a V_(H) CDR3 ofD6D3; h) a V_(H) CDR1, a V_(H) CDR2 and a V_(H) CDR3 of D1C5; i) a V_(H)CDR1, a V_(H) CDR2 and a V_(H) CDR3 of D1D1; j) a V_(H) CDR1, a V_(H)CDR2 and a V_(H) CDR3 of HB8; k) a V_(H) CDR1, a V_(H) CDR2 and a V_(H)CDR3 of HC2; l) a V_(H) CDR1, a V_(H) CDR2 and a V_(H) CDR3 of HC4; m) aV_(H) CDR1, a V_(H) CDR2 and a V_(H) CDR3 of HE3; n) a V_(H) CDR1, aV_(H) CDR2 and a V_(H) CDR3 of HF1; or o) a V_(H) CDR1, a V_(H) CDR2 anda V_(H) CDR3 of HH3.
 51. The monoclonal antibody of claim 49, whereinsaid antibody comprises: a) a V_(L) CDR1, a V_(L) CDR2 and a V_(L) CDR3of E8H11; b) a V_(L) CDR1, a V_(L) CDR2 and a V_(L) CDR3 of E8H7; c) aV_(L) CDR1, a V_(L) CDR2 and a V_(L) CDR3 of E8G12; d) a V_(L) CDR1, aV_(L) CDR2 and a V_(L) CDR3 of E8F11; e) a V_(L) CDR1, a V_(L) CDR2 anda V_(L) CDR3 of E8C9; f) a V_(L) CDR1, a V_(L) CDR2 and a V_(L) CDR3 ofD6G9; g) a V_(L) CDR1, a V_(L) CDR2 and a V_(L) CDR3 of D6D3; h) a V_(L)CDR1, a V_(L) CDR2 and a V_(L) CDR3 of D1C5; i) a V_(L) CDR1, a V_(L)CDR2 and a V_(L) CDR3 of D1D1; j) a V_(L) CDR1, a V_(L) CDR2 and a V_(L)CDR3 of HB8; k) a V_(L) CDR1, a V_(L) CDR2 and a V_(L) CDR3 of HC2; l) aV_(L) CDR1, a V_(L) CDR2 and a V_(L) CDR3 of HC4; m) a V_(L) CDR1, aV_(L) CDR2 and a V_(L) CDR3 of HE3; n) a V_(L) CDR1, a V_(L) CDR2 and aV_(L) CDR3 of HF1; or o) a V_(L) CDR1, a V_(L) CDR2 and a V_(L) CDR3 ofHH3.
 52. The monoclonal antibody of claim 44, wherein the anybody is asingle chain Fv (scFv), IgG, Fab, (Fab)₂, or (scFv')₂
 53. A compositioncomprising: a pharmaceutically acceptable carrier; and a monoclonalantibody of claim
 44. 54. The composition of claim 53, wherein saidcomposition is formulated for parenteral administration.
 55. A method oftreating a subject having cancer comprising: administering to saidsubject a therapeutically effective amount of a monoclonal antibody ofclaim
 44. 56. An isolated nucleic acid comprising a nucleotide sequenceencoding an amino acid sequence of: a V_(H) comprising a V_(H) CDR1, aV_(H) CDR2 and a V_(H) CDR3 of an antibody from clone F2-1A6; a V_(L)comprising a V_(L) CDR1, a V_(L) CDR2 and a V_(L) CDR3 of an antibodyfrom clone F2-1A6; a V_(H) comprising a V_(H) CDR1, a V_(H) CDR2 and aV_(H) CDR3 of an antibody from clone F2-1H9; a V_(L) comprising a V_(L)CDR1, a V_(L) CDR2 and a V_(L) CDR3 of an antibody from clone F2-1H9; aV_(H) comprising a V_(H) CDR1, a V_(H) CDR2 and a V_(H) CDR3 of anantibody selected from E8H11, E8H7, E8G12, E8F11, E8C9, D6G9, D6D3,D1C5, D1D1, HB8, HC2, HC4, HE3, HF1, and HH3; or a V_(L) comprising aV_(L) CDR1, a V_(L) CDR2 and a V_(L) CDR3 of an antibody selected fromE8H11, E8H7, E8G12, E8F11, E8C9, D6G9, D6D3, D1C5, D1D1, HB8, HC2, HC4,HE3, HF1, and HH3.
 57. The isolated nucleic acid of claim 56, whereinthe nucleic acid comprises a nucleotide sequence encoding an amino acidsequence of: a V_(H) comprising a V_(H) CDR1, a V_(H) CDR2 and a V_(H)CDR3 of an antibody from clone F2-1A6 and a V_(L) comprising a V_(L)CDR1, a V_(L) CDR2 and a V_(L) CDR3 of an antibody from clone F2-1A6; aV_(H) comprising a V_(H) CDR1, a V_(H) CDR2 and a V_(H) CDR3 of anantibody from clone F2-1H9 and a V_(L) comprising a V_(L) CDR1, a V_(L)CDR2 and a V_(L) CDR3 of an antibody from clone F2-1H9; or a V_(H)comprising a V_(H) CDR1, a V_(H) CDR2 and a V_(H) CDR3 of an antibodyselected from E8H11, E8H7, E8G12, E8F11, E8C9, D6G9, D6D3, D1C5, D1D1,HB8, HC2, HC4, HE3, HF1, and HH3 and a V_(L) comprising a V_(L) CDR1, aV_(L) CDR2 and a V_(L) CDR3 of an antibody selected from E8H11, E8H7,E8G12, E8F11, E8C9, D6G9, D6D3, D1C5, D1D1, HB8, HC2, HC4, HE3, HF1, andHH3.
 58. An isolated nucleic acid encoding an amino acid sequence of:(i) a V_(H) comprising a V_(H) CDR1, a V_(H) CDR2 and a V_(H) CDR3 of anantibody from clone 2D6, (ii) a V_(L) comprising a V_(L) CDR1, a V_(L)CDR2 and a V_(L) CDR3 of an antibody from clone 2D6, (iii) a V_(H)comprising a V_(H) CDR1, a V_(H) CDR2 and a V_(H) CDR3 of an antibodyfrom clone D2-1A7, (iv) a V_(L) comprising a V_(L) CDR1, a V_(L) CDR2and a V_(L) CDR3 of an antibody from clone D2-1A7, (v) a V_(H)comprising a V_(H) CDR1, a V_(H) CDR2 and a V_(H) CDR3 of an antibodyfrom clone D2-1A9, (vi) a V_(L) comprising a V_(L) CDR1, a V_(L) CDR2and a V_(L) CDR3 of an antibody from clone D2-1A9, (vii) a V_(H)comprising a V_(H) CDR1, a V_(H) CDR2 and a V_(H) CDR3 of an antibodyfrom clone D2-1B1, (viii) a V_(L) comprising a V_(L) CDR1, a V_(L) CDR2and a V_(L) CDR3 of an antibody from clone D2-1B1; (ix) a V_(H)comprising a V_(H) CDR1, a V_(H) CDR2 and a V_(H) CDR3 of an antibodyselected from A3H9, A3G3, A3D10, A3D1, A3C8, 1A3, 1A5, 1A8, 1A12, 1B2,1C2, 1C7, 1D8, and 15H11; or (x) a V_(L) comprising a V_(L) CDR1, aV_(L) CDR2 and a V_(L) CDR3 of an antibody selected from A3H9, A3G3,A3D10, A3D1, A3C8, 1A3, 1A5, 1A8, 1A12, 1B2, 1C2, 1C7, 1D8, and 15H11or; an isolated nucleic acid encoding the amino acid sequences of (i)and (ii); or (iii) and (iv); or (v) and (vi); or (vii) and (viii); or(ix) and (x).
 59. An expression vector comprising the nucleic acid ofclaim
 58. 60. A recombinant host cell comprising the vector of claim 59.61. A kit comprising a composition of claim 53 in a container.
 62. Amethod of treating a subject having cancer comprising: administering tosaid subject a therapeutically effective amount of the composition ofclaim
 53. 63. A method of detecting a cancer cell in a subjectcomprising: contacting an antibody of claim 44 with a cell of saidsubject suspected of being cancerous; and detecting said antibody boundto said cell.